Modular targeted therapeutic agents and methods of making same

ABSTRACT

Provided herein are methods for making targeted therapeutics. In several embodiments, the therapeutics are directed against soluble agents such as toxins, venoms, and/or other factors that alter physiological biopathways as well as methods of using such therapeutics to treat patients or patient populations to reduce, eliminate, or inactivate, detrimental soluble agents that such patients or patient populations have been exposed to. In several embodiments, the therapeutics are directed to patient-specific disease markers. In several embodiments, the methods comprise screening a library comprising proteins linked to their cognate mRNAs to identify mRNA-protein pairs that bind to the diseased cells, isolating one or more proteins from the identified mRNA-protein pairs, and conjugating the isolated protein(s) to a therapeutic agent.

RELATED CASES

The contents of each priority document listed in the accompanyingApplication Data Sheet is incorporated in its entirety by referenceherein. This application also incorporates by reference the sequencelisting submitted as ASCII text filed concurrently via EFS-Web. TheSequence Listing is provided as a file entitled “ST25 SequenceListing—PRONOV.009P1”, created on Mar. 1, 2013 and which is 11.4kilobytes in size.

BACKGROUND Field of the Invention

The present invention relates to methods for making therapeuticstailored to individual patients or sub-populations of patients. Severalembodiments relate to methods for making therapeutics that targetsoluble agents such as toxins, venoms, and/or other factors that alterphysiological biopathways. Methods of using such targeted therapeuticsare also provided, for example using therapeutics targeting solubleagents to treat patients or patient populations that have been exposedto such agents to reduce, eliminate, or inactivate, such detrimentalsoluble agents or their effects or use of therapeutics to treatmalignancies, pathogenic infections, and other conditions, and to reduceor prevent transplant rejection.

Description of the Related Art

Many malignant cells display epitopes that are specific not only to thetype of malignancy but also to the individual patient. Likewise, cellsthat are infected, diseased, transplanted from a donor to host, or areotherwise compromised in their health or function have been found toexpress one or more unique markers. Also, numerous toxins, venoms,chemical agents, and other agents that alter physiological biopathwaysexist and exposure to such agents presents a potentially significanthealth risk. Differentiating between the normal healthy cells of subject(or those transplanted into a subject) and those that are diseased,injured, infected or malfunctioning is important to many differenttherapies.

SUMMARY

In several embodiments, there are provided herein methods for generatinga modular targeted therapeutic that targets an agent of interest, themethod comprising: identifying a first protein capable of specificallyinteracting with the agent of interest; identifying a first mRNA thatencodes for the first protein; identifying non-antibody second portioncapable of eliciting an immune response through interaction with one ormore components of the immune system; identifying a second mRNA thatencodes for the non-antibody second portion; generating a first and asecond cDNA corresponding to each of the first and the second mRNAs;fusing the first and the second cDNA to the first and second ends of abridge cDNA to generate a fused cDNA; and translating the fused cDNAinto a corresponding fused protein, wherein a first portion of the fusedprotein is capable of interacting with the agent of interest and asecond portion of the fused protein is capable of eliciting an immuneresponse, thereby generating a bifunctional targeted therapeutic thattargets the agent of interest.

In several embodiments, the non-antibody second portion is a secondprotein. In several embodiments, the first protein is identified byscreening a library comprising proteins linked to their cognate mRNAs toidentify one or more proteins that interact with the agent of interest.Additionally, the method can optionally further comprise screening thebifunctional targeted therapeutic against the agent of interest and theone or more components of the immune system.

In several embodiments, the non-antibody second portion has a known mRNAsequence. Advantageously, this allows for generation of the encodedprotein from that mRNA, once the desired characteristics of secondprotein have been identified (e.g., interaction with desired portion ofthe immune system for the particular embodiment. For example, in severalembodiments, the second protein is capable of binding to the heavy chainof an antibody. In several embodiments, the second protein is capable ofbinding to the constant region of the heavy chain of the antibody. Inparticular, in several embodiments, the second protein is capable ofbinding to the CH1 region of the heavy chain. In particular embodiments,the non-antibody second portion is capable of binding to an IgGantibody.

In several embodiments, the agent of interest is soluble (e.g., in thebloodstream of a subject), while in additional embodiments, the agent ofinterest is a cell surface molecule or marker. In several embodiments,soluble agents of interest are selected from the group consisting ofanimal toxins, insect toxins, plant toxins, algae-derived toxins,fungi-derived toxins, bacterial-derived toxins, biowarfare agents,biopathway modulators, and combinations thereof.

In several embodiments, the agent of interest targets (and adverselyaffects) one or more of the blood, blood vessels, nervous tissue, andmuscle tissue, one or more ion channels, induces muscle paralysis,prevents blood clotting, and/or induces increased gastrointestinal watersecretion.

In several embodiments, the screening methods used to identify modulartherapeutics capable of targeting the agent of interest (out of a poolof candidate modular therapeutics) comprise positive selection, negativeselection, or combinations thereof. For example, in several embodiments,screening for bifunctional therapeutics that target the agent ofinterest comprises blocking a site on the agent of interest with whichthe first protein interacts, exposing a plurality of candidatebifunctional targeted therapeutics to the blocked agent of interest,identifying those candidate bifunctional targeted therapeutics that bindto the blocked agent of interest and those that do not bind to theblocked agent of interest; and collecting those candidate bifunctionaltargeted therapeutic that do not bind to the blocked agent of interest.In several embodiments, the collection of those that do not bind isfacilitated by first immobilizing the agent of interest on a solidsupport. Thus, a wash of the solid support and collection of the washsolution facilitates specific collection of those bifunctional targetedtherapeutics that interact with the site on the agent of interest thatwas pre-blocked.

Additionally, in several embodiments, the screening optionally furthercomprises screening the collected candidate bifunctional targetedtherapeutics for interaction with one or more components of the immunesystem. Such additional screening methods comprise, in some embodiments,exposing the collected candidate bifunctional targeted therapeutics tothe component of the immune system; identifying those candidatebifunctional targeted therapeutics that bind to component of the immunesystem and those that do not bind to the component of the immune system;and discarding those candidate bifunctional targeted therapeutics thatdo not bind to the component of the immune system and collecting thosethat do bind to the component of the immune system. As with the firstscreening described above, the component of the immune system mayoptionally be immobilized on a solid support. Likewise, the component ofthe immune system can also be pre-blocked (at a site or sites ofinterest) such that the candidate bifunctional targeted therapeuticsthat fail to bind to the blocked component are collected (as the failureto bind indicates preferential interaction with the blocked sites).

In several embodiments, the component of the immune system is anantibody. In some embodiments, the antibody is an IgG antibody. In someembodiments, the antibody is an IgG isotype 1, 2, 3, or 4 antibody.

In several embodiments there is also provided a method for treating asubject that has been exposed to a soluble agent, comprising identifyinga subject who has been exposed to a soluble agent; and administering tothe subject a bifunctional targeted therapeutic that has been screenedfor specific interaction with the soluble agent and with a component ofthe immune system, and wherein upon interaction with the soluble agentvia the first portion of the targeted therapeutic, the second portionelicits an immune response, which results in clearance of the solubleagent and treatment of the subject.

In various aspects, methods are provided herein for developing targetedtherapeutics useful in treating a wide range of conditions by targetingcell-surface markers (e.g., epitopes, idiotypes, and the like) or othermolecules that are differentially expressed by, or in close proximityto, malignant cells, pathogens, transplanted cells, and/or otherentities targeted for treatment. Also provided are methods for treatinga disease or condition by administering a therapeutic produced bymethods described herein.

In some preferred embodiments, methods provided herein utilize noveltechniques for linking proteins to their corresponding mRNAs, andscreening the protein-mRNA complexes for binding to a molecular targetassociated with one or more etiological determinants. In variouspreferred embodiments, therapeutics provided herein are designed torecognize molecular targets that are differentially expressed in anindividual patient seeking treatment, or in a sub-population ofpatients, such as patients diagnosed with a specific strain or subtypeof a disease or condition. Proteins having high affinity for a target ofinterest are preferably isolated and linked to one or more therapeuticagents effective against the disease being treated (e.g., cytotoxicagents), to produce a variety of targeted therapeutics. Advantageously,the rapid and efficient identification, isolation, and production ofproteins capable of recognizing targets of interest provides effective,low cost methods for the production of patient- and/or disease-specifictherapeutics. In various embodiments, methods provided hereinbeneficially allow a wide range of diseases and conditions to be treatedwith tailored therapeutics, within the context of existing health carebudgets and resource allocations.

In various aspects, methods are provided herein for producing tailoredtherapeutics for treating cancers and other conditions, wherein thetailored therapeutics comprise a “targeting domain” that binds to amolecular target associated with a disease or condition selected fortreatment, and a “therapeutic agent” capable of treating or preventingsaid disease or condition. In some preferred embodiments, the targetingdomain is tailored to recognize targets that are differentiallyexpressed in particular patients or sub-populations of patients, whilein these and/or other embodiments, the therapeutic agent does notrequire substantial tailoring to individual patients or sub-populationsof patients. This “modular” architecture advantageously allows for thecreation of individualized therapeutics by tailoring only the smallportion of the administrable therapeutic comprising the targetingdomain, which can then be used to enhance the efficacy of a variety ofpre-existing or easily prepared therapeutic agents.

In some preferred embodiments, the targeting domain is tailored to bindan epitope selectively or preferentially expressed by cancerous cellsrelative to non-cancerous cells in a patient seeking treatment, and thetherapeutic agent is an antibody or other molecule (hereinafter referredto as the “immune effector”) capable of stimulating an immune responsein the patient. In some preferred embodiments, the targeted epitopes aresubstantially absent from non-cancerous cells, and the therapeutic agentdoes not otherwise substantially bind to non-cancerous cells. In somepreferred embodiments, the cancer is a hematological cancer, such as alymphoma (e.g., non-Hodgkin's lymphoma), a leukemia, or a multiplemyeloma, wherein cancerous and/or malignant cells express apatient-specific epitope that can be an idiotype.

In various embodiments, the therapeutic agent is linked, fused, orderivatized, directly or indirectly, to the targeting domain to form themodular therapeutic. In some embodiments, an immune effector iscovalently linked to a target-binding domain, while in other embodimentsit is non-covalently bound. In some embodiments, the target-bindingdomain is part of a bifunctional protein comprising a target-bindingdomain fused to a second domain that binds the immune effector. Theimmune effector-binding domain may comprise an epitope recognized byvariable regions of an antibody, or a molecule that binds other regionsof an antibody, such as the Fc region. The immune effector-bindingdomain may also comprise a peptide sequence designed to bind the immuneeffector. The bifunctional protein may comprise a fusion protein, or thetwo domains can be covalently or non-covalently linked. Thetarget-binding domain and the immune effector binding domain can bedirectly linked, or indirectly linked, for example via a flexible linkerpeptide.

In other aspects, the invention provides methods for preparing atherapeutic for treating a cancer comprising isolating complexes ofexpressed mRNA molecules and their nascent polypeptides from an mRNAexpression library; screening the protein-mRNA complexes for binding toa molecular target associated with an etiological determinant, such asan epitope displayed by a cancerous cell; isolating and expressing mRNAencoding a protein that binds the target epitope; and derivatizing thetarget epitope-binding protein (or derivatives, fragments or subunitsthereof) to a therapeutic agent, such as an antibody capable ofeliciting an immune response against the target. In some embodiments,the preparation of the therapeutic further comprises allowing isolatedmRNA encoding a target-binding domain to undergo in vitro evolution,selective mutagenesis, and/or other methods known in the art to identifyand isolate mRNAs exhibiting stronger or more effective binding to thetarget epitope, as described in more detail below.

In yet further aspects, the invention provides methods for treating adisease or condition, such as a cancer, comprising identifying a targetepitope or other molecule specifically or preferentially expressed on,or in close proximity to, an etiological determinant of the conditiontargeted for treatment, in a patient in need of treatment; isolating aprotein that binds the target, but does not bind substantially tonon-targets; linking the target-binding protein (or derivatives,fragments or subunits thereof) to a therapeutic agent, such as anantibody capable of eliciting an immune response in the subject ofinterest; and administering the therapeutic to the patient in an amountand for a time sufficient to treat the condition targeted for treatment.

In a still further aspect, the invention provides methods foridentifying proteins that bind cancer cell target epitopes, or otheretiological determinants, comprising isolating complexes of expressedmRNA molecules and their nascent polypeptides from an mRNA expressionlibrary; screening the protein-mRNA complexes for binding to a targetepitope displayed by a cancerous cell; isolating and expressing mRNAencoding a protein that binds the target epitope; and derivatizing thetarget epitope-binding protein (or derivatives, fragments or subunitsthereof) to an antibody capable of eliciting an immune response. In someembodiments, the methods may further comprise allowing isolated mRNAencoding a target epitope-binding protein to undergo in vitro evolution,selective mutagenesis, or other methods known in the art to identify andisolate mRNAs exhibiting stronger or more effective binding to thetarget epitope.

In yet another aspect, the invention provides a kit for developing anindividualized therapeutic for the treatment of a disease or conditioncharacterized by the expression of disease- and/or patient-specificetiological determinants. In some preferred embodiments, a kit isprovided for developing patient-specific therapeutics for the treatmentof a cancer, including solid tumors and hematological cancers, whereinthe therapeutics are targeted to a unique cell-surface epitopedifferentially expressed on the surface of cancerous and/or malignantcells.

In an additional aspect, the invention provides therapeutics and methodsfor reducing or preventing transplant rejection. In some embodiments,the target-binding protein binds to cell surface antigens displayed bytransplanted cells, such as MHC antigens, and the therapeutic agent(e.g., the immune effector) comprises a protein or other molecule thatbinds to and inhibits one or more molecular determinants of the immuneresponse. In some preferred embodiments, the immune effector binds theC1 q or C3 components of the complement system to thereby inhibit theactivation of complement-mediated immunity. In other embodiments, theimmune effector stimulates an immune response to eliminate transplantedcells bearing “foreign” MHC or other antigens. In further embodiments,the therapeutic agent ablates or prevents the clonal expansion oflymphocyte subpopulations expressing specific epitopes, for exampleepitopes that recognize MHC antigens.

Several embodiments of the present invention further provide methods andkits for inhibiting transplant rejection that are essentially similar tothose described above for treating malignancies.

Further aspects of the invention provide therapeutics, methods and kits,essentially similar to those described above, wherein the target-bindingdomain is selected to bind one or more variable epitopes expressed by adisease-causing pathogen, or one or more cells infected by adisease-causing pathogen, and the therapeutic agent comprises ananti-pathogenic drug, such as an antibiotic or other cytotoxic agent. Insome embodiments, the pathogen is a virus, such as HIV.

In various aspects, methods provided herein allow for the rapid andcost-effective creation of individualized therapeutics. The followingdetailed description illustrates various aspects of the invention asthey relate to particular applications. However, the description appliesequally to the development and use of therapeutics and methods for thetreatment of a wide variety of conditions, including but not limited to,the treatment of malignancies, the reduction and elimination ofpathogens, the reduction and/or prevention of transplant rejection, andin general, the treatment of any condition involving a therapeutictarget which exhibits differential binding characteristics relative tonon-targeted cells or molecules.

Additionally, in various aspects, methods are provided herein fordeveloping targeted therapeutics useful in treating a wide range ofconditions by targeting soluble targets (e.g., toxins, venoms and thelike). Methods are also provided herein that target soluble proteinswithout a pre-immunization of a subject with an epitope of the desiredtarget. Also provided are methods for treating a disease or condition byadministering a therapeutic produced by methods described herein.

In several embodiments, there is provided a method for generation of abifunctional targeted therapeutic that targets a soluble agent, themethod comprising identifying a first protein capable of interactingwith a desired soluble target, identifying a first mRNA that encodes forthe first protein, identifying an antigen capable of eliciting an immuneresponse through interaction with one or more components of the immunesystem, identifying a second mRNA that encodes for the antigen,generating a first and a second cDNA corresponding to each of the firstand the second mRNAs, fusing the first and the cDNA to generate a fusedcDNA, translating the fused cDNA into a corresponding fused protein,wherein the a first portion of the fused protein is capable ofinteracting with the desired soluble target and a second portion of thefused protein is capable of eliciting an immune response, therebygenerating a bifunctional targeted therapeutic that targets a solubleagent. In one embodiment, the antigen has a known mRNA sequence.

In several embodiments, there is provided a method for generation of abifunctional targeted therapeutic that targets a soluble agent, themethod comprising identifying a first protein capable of interactingwith a desired soluble target, identifying a first mRNA that encodes forthe first protein, identifying a second protein capable of eliciting animmune response through interaction with one or more components of theimmune system, identifying a second mRNA that encodes for the secondprotein, generating a first and a second cDNA corresponding to each ofthe first and the second mRNAs, fusing the first and the cDNA togenerate a fused cDNA, translating the fused cDNA into a correspondingfused protein, wherein the a first portion of the fused protein iscapable of interacting with the desired soluble target and a secondportion of the fused protein is capable of eliciting an immune response,thereby generating a bifunctional targeted therapeutic that targets asoluble agent. In some embodiments, the second protein is capable ofbinding to an antibody. In some embodiments, the second protein iscapable of binding to the heavy chain of an antibody. In someembodiments, the second protein is capable of binding to the constantregion of the heavy chain. In some embodiments, the second protein iscapable of binding to the CH1 region of the heavy chain. In someembodiments, the antibody is an IgG antibody.

In several embodiments, the proteins are linked to their cognate mRNAsvia a cross-linker. In one embodiment, the cross-linker is placed on acodon. In one embodiment, the cross-linker is placed on a pseudo-stopcodon. In one embodiment, the cross-linker comprises a psoralencross-linker, and wherein exposure of the mRNA to UV light links themRNA to the protein. In some embodiments, the linker is selected fromthe group consisting of tRNA, modified tRNA, and tRNA analogs.

In some embodiments, the first protein is identified by screening alibrary comprising proteins linked to their cognate mRNAs to identifyone or more proteins that interact with the soluble agent. In someembodiments, the method further comprises screening the bifunctionaltargeted therapeutic against the soluble agent and the one or morecomponents of the immune system. In some embodiments, the fused cDNAcomprises a bridge cDNA between the first and the second cDNA.

In some embodiments, the soluble agent is a selected from the groupconsisting of animal toxins, insect toxins, plant toxins, algae-derivedtoxins, fungi-derived toxins, bacterial-derived toxins, biowarfareagents, and biopathway modulators.

In some embodiments, the soluble agent targets one or more of the blood,blood vessels, nervous tissue, and muscle tissue.

In some embodiments, the soluble agent targets an ion channel.

In some embodiments, the soluble agent induces muscle paralysis.

In some embodiments, the soluble agent targets prevents blood clotting.

In some embodiments, the soluble agent induces increasedgastrointestinal water secretion.

In several embodiments, there is provided a method for treating asubject that has been exposed to a soluble agent, comprising:identifying a subject who has been exposed to a soluble agent; andadministering to the subject a bifunctional targeted therapeutic,wherein the immune response results in clearance of the soluble agent.

In several embodiments, there is provided a method for treating asubject that has been exposed to a soluble agent, comprising identifyinga subject who has been exposed to a soluble agent, administering to thesubject an antigen, wherein administration of the antigen inducesproduction of antibodies directed to the antigen by the subject;administering to the subject the bifunctional targeted therapeuticcomprising the antigen, wherein the administration allows the targetedtherapeutic to bind to interact with the soluble agent and with theproduced antibodies, wherein the interaction results in clearance thesoluble target by the immune system, thereby treating the subject.

In several embodiments, there is provided a method for treating asubject that has been exposed to a soluble agent, comprising identifyinga subject who has been exposed to a soluble agent, administering to thesubject an antigen in order for the subject to produce antibodies to theantigen, identifying a first protein capable of interacting with thesoluble agent, identifying a first mRNA that encodes for the firstprotein, identifying a second mRNA that encodes for the antigen,generating a first and a second cDNA corresponding to each of the firstand the second mRNAs, fusing the first and the cDNA to generate a fusedcDNA, translating the fused cDNA into a corresponding fused protein,administering the fused protein to the subject, wherein theadministration allows the first portion of allows a first portion of thefused protein to interact with the soluble agent and a second portion ofthe fused protein to interact with the antibodies produced in responseto the administration of the antigen, wherein the interactions result inthe clearance of the soluble agent by the immune system, therebytreating the subject.

In several embodiments there is provided a method for treating a subjectthat has been exposed to a soluble agent, comprising, identifying asubject who has been exposed to a soluble agent, identifying a firstprotein capable of interacting with the soluble agent, identifying afirst mRNA that encodes for the first protein, identifying a secondprotein capable of binding to an antibody, identifying a second mRNAthat encodes for the second protein, generating a first and a secondcDNA corresponding to each of the first and the second mRNAs, fusing thefirst and the cDNA to generate a fused cDNA, translating the fused cDNAinto a corresponding fused protein, administering the fused protein tothe subject, wherein the administration allows a first portion of thefused protein to interact with the soluble agent and a second portion ofthe fused protein to interact with the antibody, wherein the interactionwith the antibody elicits an immune response, and wherein the immuneresponse results in clearance of the soluble agent, thereby treating thesubject.

In one embodiment, the antibody is an IgG antibody.

In several embodiments there is provided a use of bifunctionaltherapeutic generated according to the methods disclosed herein for thetreatment of exposure to a soluble agent, wherein the exposure induces adeleterious effect in an exposed subject, and wherein the bifunctionaltherapeutic clears the soluble agent from the subject, thereby treatingthe exposure.

In several embodiments there is provided a use of a bifunctionaltherapeutic comprising a known antigen for the pre-treatment of asubject likely to be exposed to a soluble agent, wherein administrationto the identified antigen induces the production of antibodies directedagainst the antigen, wherein subsequent actual exposure to the solubleagent induces the interaction of the first portion of the bifunctionalprotein with the soluble agent and the interaction of the producedantibodies with the second portion of the bifunctional protein, andwherein the interactions clear the soluble agent from the subject.

In several embodiments, methods provided herein utilize novel techniquesfor linking proteins to their corresponding mRNAs, and screening theprotein-mRNA complexes for binding to a target associated with aparticular soluble agent. In some embodiments, the identified proteinshaving high affinity for a soluble agent of interest are preferablyisolated and linked to one or more immune modulators, to produce avariety of targeted therapeutics. Advantageously, the rapid andefficient identification, isolation, and production of proteins capableof recognizing targets of interest provides effective, low cost methodsfor the production of patient- and/or condition-specific therapeutics.In various embodiments, methods provided herein beneficially allow awide range of soluble agents, and exposure thereto, to be treated withtailored therapeutics, within the context of existing health carebudgets and resource allocations.

In various aspects, methods are provided herein for producing fortreating exposure, or the possible exposure in the future to certaindeleterious soluble agents, the therapeutics comprise a “targetingdomain” that binds to all or a portion of a soluble agent, and a immuneeffector region capable of initiating an immune response. This “modular”architecture advantageously allows for the creation of uniquely targetedtherapeutics by tailoring the targeting domain, which can then be usedto enhance the efficacy of a variety of pre-existing or easily preparedtherapeutic agents.

In various embodiments, the immune effector region of the therapeutic islinked, fused, or derivatized, directly or indirectly, to the solubleagent targeting domain to form the bifunctional therapeutic. In someembodiments, an immune effector is covalently linked to a target-bindingdomain, while in other embodiments it is non-covalently bound. Inseveral embodiments, the target-binding domain and the immune effectorbinding domain can be directly linked, or indirectly linked, for examplevia a flexible linker peptide.

In other aspects, the invention provides methods for preparing atherapeutic for treating exposure to a soluble agent comprisingisolating complexes of expressed mRNA molecules and their nascentpolypeptides from an mRNA expression library; screening the protein-mRNAcomplexes for binding to all or a portion of a soluble agent; isolatingand expressing mRNA encoding a protein that binds the target; andlinking the target-binding protein (or derivatives, fragments orsubunits thereof) to a therapeutic agent, such as an antibody capable ofeliciting an immune response. In some embodiments, the preparation ofthe therapeutic further comprises allowing isolated mRNA encoding atarget-binding domain to undergo in vitro evolution, selectivemutagenesis, and/or other methods known in the art to identify andisolate mRNAs exhibiting stronger or more effective binding to thetarget, as described in more detail below.

In yet another aspect, the invention provides a kit for developing abifunctional therapeutic for the treatment of (or pre-treatment of)exposure to a soluble agent. In some embodiments, a kit is provided fordeveloping patient-specific therapeutics for the treatment of solubleagent exposure, wherein the therapeutics are targeted to a uniquemarker, epitope, structural feature, etc., that is differentiallyexpressed by the soluble agent (as compared to other molecules orcompounds in the bloodstream.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a complex comprising a target epitope-binding protein andan immune effector antibody reacted with a malignant cell via the Fabidiotype expressed on the cell surface.

FIG. 2 depicts an illustration of a known human antibody (Ab) to a smallhuman protein (Ebbd). The protein can serve as an epitope that links theantibody (Ab) to the therapeutic. The role of the Ab is to act as animmune effector that ultimately triggers an immune response.

FIG. 3 depicts an illustration of scaled up production of mRNA encodingan immune effector-binding protein (Ebbd) for ligation purposes inconstruction of oligonucleotide sequence that codes for the bifunctionalprotein component of the therapeutic.

FIG. 4 depicts a system for breeding a protein that binds selectively toan epitopes expressed on the surface of certain cancer cells. The systemincludes in vitro translation of an mRNA library to give proteinlibraries in which each protein remains linked with its cognate mRNA.Selection includes steps to identity proteins that selectively bind thecancer epitopes but not the epitopes expressed by normal cells. Theselection methods used are dependent on the epitopes isolation anddisplay procedure used. The system also includes rapid directedevolution, selection, and production, in quantity, of the protein(s)with targeted properties.

FIG. 5 depicts one embodiment of a process for isolating the mRNA fromthe protein that binds the idiotype or target epitope on malignantcells. The illustration excludes negative selection.

FIG. 6 depicts one embodiment of a bifunctional protein translated froman oligonucleotide sequence created by the ligation of the mRNAs thatcode for a target epitope-binding protein and for a human protein (theimmune effector binding domain (Ebbd)) that is bound by a humanantibody.

FIG. 7 depicts an individualized cancer therapeutic prepared by forminga complex that includes a bifunctional protein with epitopes for theimmune effector Ab (Anti-Ebbd antibody) and the Fab idiotype or targetepitope.

FIG. 8 depicts an illustration of a malignant cell tagged by thetherapeutic through the linkage between the idiotype or target epitopeand the protein that binds the idiotype or target epitope. The exposedhuman antibody triggers the curative immune response.

FIG. 9 depicts a therapeutic comprising a first peptide (the targetepitope-binding domain) bred to bind to the target epitope and a secondpeptide (the immune effector-binding domain) bred to bind a stable C3convertase (the immune effector). Binding of the therapeutic to thetarget epitope elicits a complement-mediated immune response at thebinding site.

FIG. 10 depicts a therapeutic comprising a first peptide (the targetepitope-binding domain) bred to bind to the target epitope and a secondpeptide (the immune effector) bred to bind the C1 component of thecomplement system. Binding of the therapeutic to the target epitopeelicits binding of endogenous C1 to the effector, and the initiation ofa complement-mediated immune response at the binding site.

FIG. 11 illustrates schematically one example of the complex formed bythe mRNA and its protein product when linked by a modified tRNA oranalog. As shown, a codon of the mRNA pairs with the anticodon of amodified tRNA and is covalently crosslinked to a psoralen monoadduct, ora non-psoralen crosslinker or aryl azides by UV irradiation. Thetranslated polypeptide is linked to the modified tRNA via the ribosomalpeptidyl transferase. Both linkages occur while the mRNA and nascentprotein are held in place by the ribosome.

FIG. 12 illustrates schematically an example of the in vitro selectionand evolution process, wherein the starting nucleic acids and theirprotein products are linked (e.g., according to FIG. 1 ) and areselected by a particular characteristic exhibited by the protein.Proteins not exhibiting the particular characteristic are discarded andthose having the characteristic are amplified with variation, preferablyvia amplification with variation of the mRNA, to form a new population.In various embodiments, nonbinding proteins will be selected. The newpopulation is translated and linked via a modified tRNA or analog, andthe selection process is repeated. As many selection andamplification/mutation rounds as desired can be performed to optimizethe protein product.

FIG. 13 illustrates one method of construction of a tRNA molecule of theinvention. In this embodiment, the 5′ end of a tRNA, a nucleic acidencoding an anticodon loop and having a molecule capable of stablylinking to mRNA (such as psoralen, as used in this example), and the 3′end of tRNA modified with a terminal puromycin molecule are ligated toform a complete modified tRNA for use in the in vitro evolution methodsof the invention. Other embodiments do not include puromycin.

FIG. 14 describes two alternative embodiments by which the crosslinkingmolecule psoralen can be positioned such that it is capable of linkingthe mRNA with the tRNA in the methods of the invention. A firstembodiment includes linking the crosslinker (e.g., psoralen monoadduct)to the mRNA, and a second embodiment includes linking the crosslinker tothe anticodon of the tRNA molecule. The crosslinker can either bemonoadducted to the anticodon or the 3′ terminal codon of the readingframe for known or partially known messages. This can be done in aseparate procedure from translation, e.g., before translation occurs.

FIG. 15 illustrates the chemical structures for uridine andpseudouridine. Pseudouridine is a naturally occurring base found in tRNAthat forms hydrogen bonds just as uridine does, but lacks the 5-6 doublebond that is the target for psoralen.

FIG. 16 illustrates some embodiments of the present invention. The SATA,Linking tRNA Analog and Nonsense Suppressor analog, in certainembodiments, are shown.

FIGS. 17A-17D depict a scheme for development of therapeutics targetedto soluble targets. Panel A depicts identification of an mRNA linked toits cognate protein. A soluble target (e.g., a toxin or venom) on solidsubstrate is panned with a library of proteins (each of which may or maynot interact with the soluble target) that are linked to their cognatemRNA. Panel B depicts identification of an mRNA associated with a knownantigen, that antigen to be used to immunize a patient. Panel C depictsthe fusion of the cDNAs from the mRNAs identified in each of Panels Aand B. Panel D depicts the generation of a pool of bifunctional proteinsdirected to the soluble target, which are generated through translationof the cDNA with a linker provided between the two ends of the bifunctional protein.

FIGS. 18A-18D depict a scheme for selecting bifunctional proteins thatspecifically react with the soluble target of interest and the knownantigen used to immunize a subject. Panel A depicts the panning of thepool of bifunctional proteins against the soluble target which isaffixed to a solid substrate. Panel B depicts specific binding ofcertain bifunctional proteins to the target of interest. Panel C depictsthe reaction of the antigen portion of the bifunctional protein with aspecific antibody bound to a substrate. Panel D represents the pool ofgenerated bifunctional proteins that are to be used in treatments (e.g.,those that have both a portion that reacts with the soluble target ofinterest and the known antigen of interest).

FIGS. 19A-19C depict a schematic for the treatment of a subject who hasbeen exposed to the soluble target. Panel 3A depicts the administrationof the targeted therapeutic to the subject. Panel B represents the invivo activity of the targeted therapeutic, e.g., the soluble target isbound to one end of the therapeutic and the other end of the therapeuticis bound by an antibody present in the subject. Panel C depicts thecomplex that is formed, which is then destroyed by the immune system.

FIGS. 20A-20D depict a schematic for development of therapeuticstargeted to soluble targets to be administered to subjects who have notbeen pre-immunized with a known antigen. Panel A depicts identificationof a first mRNA linked to its cognate protein. A soluble target (e.g., atoxin or venom) on solid substrate is panned with a library of proteins(each of which may interact with the soluble target) that are linked totheir cognate mRNA. Panel B depicts an IgG molecule on a solid substratethat is panned with the library of proteins that are linked to theircognate mRNA, thereby identifying those proteins that react with the CH1region of the antibody. Panel C depicts the generation of a fusionprotein from cDNAs generated from each of the identified mRNAs. Panel Drepresents the pool of bifunctional proteins that are to be used intreatments (e.g., those that have both a portion that reacts with thesoluble target of interest and the CH1 region of an antibody).

FIG. 21 depicts an example of the therapeutic agent generated by methodsdescribed herein.

FIGS. 22A-22D depicts a scheme for selecting bifunctional proteins thatspecifically react with the soluble target of interest and the CH1region of an antibody. Panel A depicts the panning of the pool ofbifunctional proteins against the soluble target which is affixed to asolid substrate. Panel B depicts specific binding of certainbifunctional proteins to the target of interest. Panel C depicts thereaction of the bifunctional protein with the CH1 region of antibodiesbound to a substrate. Panel D represents the pool of generatedbifunctional proteins that are to be used in treatments (e.g., thosethat have both a portion that reacts with the soluble target of interestand with the CH1 region of an antibody).

FIGS. 23A-23E depict the process in vivo that results in the clearanceof the soluble target and generation of cells bearing antigen related tothe soluble target. Panel A depicts a typical macrophage having an Fcreceptor on its surface as well as the soluble target-bifunctionalprotein-antibody complex. Panel B depicts the binding of the complex tothe macrophage. Panel C depicts the phagocytosis of the complex by themacrophage. Panel D depicts the digestion of the complex into smallpeptide fragments by enzymes. Panel E depicts the generation of amacrophage with the small peptides expressed on its surface, which willinduce antibody production.

DETAILED DESCRIPTION

As used herein, the terms “bifunctional therapeutic”, “targetedtherapeutic”, and “tailored therapeutic” shall be used interchangeably,shall be given their ordinary meaning and shall also refer totherapeutics with a soluble agent targeting portion and an immuneeffector portion (e.g., an antigen or an antibody binding region).

The terms “T lymphocyte” and “T cell” as used herein shall be giventheir ordinary meaning and shall also encompass any cell within the Tlymphocyte lineage from T cell precursors to mature T cells.

The terms “B lymphocyte” and “B cell” shall be given their ordinarymeaning and shall also encompass any cell within the B cell lineage fromB cell precursors, such as pre-B cells, to mature B cells and plasmacells.

Immunoglobulin molecules consist of heavy (H) and light (L) chains,which comprise highly specific variable regions at their amino termini.The variable (V) regions of the H (V_(H)) and L (V_(L)) chains combineto form the unique antigen recognition or antigen combining site of theimmunoglobulin (Ig) protein. The variable regions of an Ig moleculecontain determinants (e.g., molecular shapes) that can be recognized asantigens or idiotypes.

The term “epitope” shall be given its ordinary meaning and shall alsorefer to the set of antigenic or epitopic determinants (i.e., idiotopes)of an immunoglobulin V domain (i.e., the antigen combining site formedby the association of the complementarity determining regions or V_(H)and V_(L) regions).

The term “idiotope” shall be given its ordinary meaning and shall alsorefer to a single idiotypic epitope located along a portion of the Vregion of an immunoglobulin molecule.

The term “immune effector” shall be given its ordinary meaning and shallalso refer to a molecule, or derivatives, fragments, or subunitsthereof, able to stimulate an immune response in the subject beingtreated, and may comprise an antibody, or derivatives, fragments, orsubunits thereof, or a non-antibody molecule.

An “adjuvant” shall be given its ordinary meaning and shall also referto a compound which enhances or stimulates the immune response whenadministered with one or more antigen(s).

The term “malignant cells” shall be given its ordinary meaning and shallalso refer to cells, which if left untreated, give rise to a cancer.

The terms “protein,” “peptide,” and “polypeptide” shall be given theirordinary meaning and shall also refer to a polymeric molecule of two ormore units comprised of amino acids in any form (e.g., D- or L- aminoacids, synthetic or modified amino acids capable of polymerizing viapeptide bonds, etc.), and these terms may be used interchangeablyherein.

The terms “soluble target” and “soluble agent” shall be given theirordinary meaning and shall also refer to toxins, venoms, factors thathave the capacity to alter biochemical pathways, biochemical agents, andthe like that are not solid or tissue-based (e.g., they present in theblood circulation of a subject as opposed to being a mass of cells,etc.). Non-limiting examples of soluble targets are shown in Tables 1-8,below.

Limited therapies are available for subjects exposed to certain solublemolecules, (e.g., toxins). For example, antivenin against the venomparticular snakes can be used to treat snake bites. The principle ofantivenin is based on that of vaccines. Rather than inducing immunity inthe patient directly, it is induced in a host animal and thehyperimmunized serum is transfused into the patient. Unfortunately, thenumber and variety of soluble toxins and agents is greater than thecurrent host of therapies available to treat them.

Thus, there exists a need for methods of producing therapeutics that arespecifically targeted against specific soluble targets and for methodsof treating individuals or populations that have been exposed to suchagents.

Provided herein, are methods for producing targeted therapeutics thatare tailored to specific soluble targets, as well as methods fortreating subjects who have been exposed to such soluble targets. Inseveral embodiments, the therapeutics are comprised of a “modular”architecture that allows a portion of the therapeutic to bind, engage,or otherwise interact the soluble target, and a second domain thatbinds, engages, or otherwise interacts with one or more components of asubject's immune system.

Lymphocytes are critical to the immune system of vertebrates.Lymphocytes are produced in the thymus, spleen and bone marrow (adult)and represent about 30% of the total white blood cells present in thecirculatory system of humans (adult). There are two majorsub-populations of lymphocytes: T cells and B cells. T cells areresponsible for cell-mediated immunity, while B cells are responsiblefor antibody production (humoral immunity). In a typical immuneresponse, T cells are activated when the T cell receptor binds tofragments of an antigen that are bound to major histocompatibilitycomplex (“MHC”) glycoproteins on the surface of an antigen presentingcell; such activation causes release of biological mediators(“interleukins”) which, in essence, stimulate B cells to differentiateand produce antibody (“immunoglobulins”) against the antigen.

The etiology of hematological cancers such as lymphomas, leukemias andmultiple myelomas varies or is unknown. Suspected causes range fromviral and chemical exposure to familial propensities. A commondenominator in these cancers however, is that they all begin with amalignantly transformed B-cell or T-cell which divides to form a cloneof cells that express the same Fab idiotype on the immunoglobulinproteins they express on their surface. One of the difficulties intreating these cancers is that each cancer expresses a unique idiotype.Developing a therapeutic treatment that effectively and selectivelytreats all possible idiotypes has therefore been elusive.

Conventional treatments for hematological cancers typically involveprocedures that destroy all blood producing cells in the bone marrow,including the malignant clone, followed by bone marrow replacement withstem cells isolated from the patient or bone marrow from a matched donorto reconstruct the blood producing system. These treatments are highlyinvasive and marginally curative. One approach involves treatment withmonoclonal antibody vaccines that recognize cell surface proteinsutilized as “markers” for identification. Therapeutics adopting thisapproach include Compath-H (Alemtuzumab), HLL2 (Epartuzumab), Hu1D10,and Rituximab, (e.g., U.S. Pat. No. 6,455,043). However, a seriouslimitation with these monoclonal antibody based therapeutics is that thetargeted cell surface antigens are often found on both normal as well asmalignant cells. In addition, because of the difficulties in producinghuman monoclonal antibodies, monoclonal antibody vaccines typicallyutilize “Chimeric” antibodies, i.e., antibodies which comprise portionsfrom two or more different species (e.g., mouse and human). Repeatedinjections of such foreign antibodies can lead to the induction ofimmune responses leading to harmful hypersensitivity reactions. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody response (“HAMA” response). Patients may alsodevelop a Human Anti-Chimeric Antibody response (“HACA” response). HAMAand HACA can attack “foreign” antibodies so that they are, in effect,neutralized before they reach their target site(s). A further drawbackto monoclonal antibody vaccines is the time and expense required toproduce monoclonal antibodies. This is particularly problematicconsidering that targeted epitopes, such as CD20, CD19, CD52w, andanti-class II HLA can readily mutate to form new tumors that areresistant to previous therapeutics (see e.g., Clinical Cancer Research,5:611-615, 1999). Thus, there is a need in the art for effective, lowcost therapeutics for treating malignancies by selectively targeting anindividual's cancerous cells over benign cells.

Like malignant cells, the cells of transplanted tissues and organsdisplay cell-surface epitopes that are differentially expressed intransplanted cells relative to native cells. In some aspects, thepresent invention is directed to therapeutics that can be targeted tothe cells of transplanted tissues or organs by recognition of suchvariable epitopes. Transplant rejection is caused by an immune responseto alloantigens on the transplanted cells, which are proteins specificfor an individual patient (including the donor), and which are thusperceived as foreign by the recipient. The most common alloantigensinvolved in transplant rejections are MEW (major histocompatibilitycomplex) molecules, which are expressed on the surface of transplantedcells and are highly polymorphic among individuals. Foreign MHCmolecules are recognized by the recipient's immune system, causing animmune response that leads to rejection of the transplant.

One pathway through which the immune system rejects transplanted tissuesis complement-mediated immunity, which can be activated by binding of C1(a first component of complement) to an immune complex consisting of anMHC antigen on a transplanted cell and the recipient's natural antibodyagainst the MHC antigen. Activation of the pathway results in theassembly of enzymes called C3 convertases, which cleave the complementcomponent C3 to form C3 a and C3 b. Some of the C3 b molecules then bindto the C3 convertases to cleave C5 to C5 a and C5 b. The biologicalactivities of the complement system, in turn, are derived from thecleavage products of C3 and C5. Another subcomponent of the complementsystem, C1 q, is involved in the initial steps of complement activation.To date, methods for treating transplant rejection by modulatingcomplement-mediated immunity have suffered from side effects associatedwith non-selectivity, due, for example, to the suppression of allcomplement-mediated immune responses by a therapeutic agent, whicheliminates an important component of the immune system's ability toprotect against foreign molecules.

In some aspects, several embodiments of the present invention aredirected to therapeutics that can be targeted to patient-specificepitopes, such as, for example those displayed on malignant lymphocytes,infected tissues, or tissues expressing unique disease markers.

Likewise, several embodiments provide therapeutics for reducing orpreventing transplant rejection by selectively inhibiting the body'simmune response against transplanted cells while retaining protectionagainst foreign pathogens, and/or by selectively destroying particularcell types that stimulate a larger immune response.

Additionally, provided herein, are methods for producing individualizedtherapeutics that are tailored to specific patients, or tosub-populations of patients suffering from a particular disease orcondition. In various embodiments, the therapeutics are comprised of a“modular” architecture that allows tailoring of a small protein domain(the “target-binding domain”) to bind one or more patient- and/ordisease-specific markers, and use of the tailored domain to direct avariety of existing or easily produced therapeutics that are effectiveagainst the condition targeted for treatment. The targeted diseasemarkers can comprise any type of molecule, or portion or derivativethereof, or complex of molecules, including but not limited to,proteins, nucleic acids, lipids, chemical compounds, polymers, andmetals, as well as biological structures, such as cell membranes,cytoskeletal elements, receptors, and even entire cells. Advantageously,the markers are expressed on, or in close proximity to, an etiologicaldeterminant of the condition targeted for treatment, and activity of thetherapeutic agent is focused to disease-causing cells, pathogens,proteins, and/or other determinants of the condition being treated.

In some embodiments, the modular therapeutic is tailored for thetreatment of a hematological cancer, and designed to bind to the uniqueFab idiotype on the surface of malignant B-cells and/or T-cells. Forexample, in some preferred embodiments, a therapeutic is provided forthe treatment of non-Hodgkin's Lymphoma (NHL), which is a “clonal”B-cell disease, in which all cancerous cells are derived from a single,malignant B-cell. As a result, every NHL cell expresses a commonidiotype (comprising the variable domains of surface expressed IgMmolecules) that is unique to each patient, which can be targeted by thetarget-binding portions of the modular therapeutics provided herein.B-cells can be isolated from the lymph nodes, or from peripheral blood,using methods known in the art. For example, in some embodiments,erythrocytes and/or granulocytes may be separated from the B-cells bycentrifugation in a liquid having a density intermediate between thegroups of cells to be separated. Means of obtaining T-lymphocytes arealso well known in the art, such as isolation from the peripheral bloodof a patient, and separation on the basis of size and/or density.Extraction of proteins from B-cells and/or T-cells may be performed byany of the many means known in the art. For example, cells may be lysedby a detergent or by mechanical means. If desired, nucleic acids can beremoved from the cell preparation by enzymatic digestion or byprecipitation with agents such as streptomycin. Such means are wellknown in the art.

The mechanism of action of a modular therapeutic for treating ahematological cancer prepared according to one embodiment of the methodsdescribed herein is illustrated in FIG. 1 . The administered therapeuticbinds to the unique Fab idiotype on the surface of malignant B-cellsand/or T-cells, and the etiological determinant of the hematologicalcancer, with which the idiotype is associated, comprises malignantand/or cancerous cells expressing the targeted idiotype. Binding of thetargeting domain to the idiotype effects an immune response thatdestroys the malignant cells. By itself, the target-binding protein istoo small to elicit an immune response, and thus the bound immuneeffector will not produce an IR in the absence of binding to thetargeted idiotype. When the target-binding protein binds to the Fabidiotype on the surface of a malignant cell, the malignant cell acts asa carrier that confers immunogenicity to the target epitope-bindingprotein, allowing the immune effector to produce an IR that targets themalignant cell. In further embodiments, therapeutics and methods of theinvention can also be used to target epitopes that distinguish nonhematological cancers.

In various preferred embodiments, targeted markers are differentiallyexpressed (i) in an individual patient, or a defined sub-group ofpatients, relative to other patients having similar diagnoses, and/or(ii) in association with cells or other molecular targets associatedwith the etiology of the condition, relative to cells/molecules that areunassociated with the condition and preferably not subjected to thetherapeutic agent. Advantageously, the selectivity of tailoredtherapeutics enhances the efficacy of treatment relative to existing,non-tailored therapeutics, due, for example, to the non-selectiveactivity of non-tailored therapeutics against healthy cells, and/orfailure of existing targeted therapeutics to account for inter-patientvariability in the targeted marker(s). For example, in the case ofhematological cancers, the targeted idiotype is unique to both thepatient and to the malignant cells, allowing the activity of thetherapeutic agent (effecting an immune response) to be selectivelydirected to targeted cells, sparing non-cancerous cells. Moreover,because the idiotype is unique to each patient, non-tailoredtherapeutics would result in a non-selective, or less-selective,therapeutic response.

Examples of cell-surface markers differentially expressed by malignantcells include, but are not limited to: for fluid tumors, stable cellsurface antigen epitopes, such as CD-20 and CD-22, and for solid tumors,surface epitopes, such as CD-19 and CD-33, which become internalizedupon binding with a mAb. Other differentially expressed cell-surfacemarkers are known in the art, including but not limited to, CD-52w andclass II HLA antigens. In some preferred embodiments, the targetedepitope is a cancer cell-specific epitope that is mutated (see e.g.,Clinical Cancer Research, 5:611-615, 1999) in the patient targeted fortreatment. Advantageously, methods provided herein allow for moreefficacious treatment of cancers by allowing the development ofindividualized therapeutics that target mutated epitopes, which areunique to each patient.

In some preferred embodiments (as shown, e.g., in FIG. 2 ), thetarget-binding domain of the modular therapeutic comprises a firstsub-domain that binds the target (e.g., a target epitope-binding domain(Tebd)), and a second sub-domain that binds the therapeutic agent (e.g.,an immune effector-binding domain (Ebbd)). In some embodiments, thetherapeutic agent is an agent that is capable of stimulating an immuneresponse in the subject targeted for treatment, such as an antibody, ora derivative, fragment, or subunit thereof, and the domain that bindsthe therapeutic agent is a small protein recognized by the therapeuticagent, for example an epitope recognized by a therapeutic agentantibody. Well-characterized antibody-antigen pairs can also be utilizedthat are known in the art, and are commercially available. Antibodiesuseful in the invention may be derived from any mammal, or may be achimeric antibody derived from a combination of different mammals. Themammal may be, for example, a rabbit, a mouse, a rat, a goat, or ahuman. The antibody is preferably a human antibody. Reactivity ofantibodies against a target antigen may be established by a number ofwell known means, including Western blot, immunoprecipitation, ELISA,and FACS analyses using, as appropriate, Fab idiotype fragments,peptides, idiotype-expressing cells or extracts thereof. The antibodycan belong to any antibody class and/or sub-class. The antibodies mayalso contain fragments from antibodies of different classes andsub-classes, thereby forming a composite.

In various embodiments, human monoclonal antibodies having a desiredbinding activity are produced using methods known in the art (forreview, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539), forexample by screening a phage display library, as described, e.g., inParmley and Smith Gene 73:305-318 (1988), Barbas et al., Proc. Natl.Acad. Sci. USA 88: 7978-7982 (1991), Griffiths et al., EMBO J 13:3245-3260 (1994), Griffiths and Hoogenboom, Building an in vitro immunesystem: human antibodies from phage display libraries. In: ProteinEngineering of Antibody Molecules for Prophylactic and TherapeuticApplications in Man. Clark, M. (Ed.), Nottingham Academic, pp 45-64(1993), and Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82, all of which are herein incorporated byreference. Typically, clones corresponding to antibodies which producebinding affinities of a desired magnitude are identified, and the DNA isused to produce the antibodies of interest using standard recombinantexpression methods.

Fully human monoclonal antibodies may also be produced using transgenicmice engineered to contain human immunoglobulin gene loci, as describedin PCT Patent Application WO98/24893 and Jakobovits, 1998, Exp. Opin.Invest. Drugs 7(4): 607-614, herein incorporated by reference. Thismethod avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

In some embodiments, an antibody against an antigen of interest, such asan immune effector-binding domain, is produced in the patient selectedfor treatment with the modular therapeutic. For example, in someembodiments, the patient is “vaccinated” with the antigen of interest,and antibodies from the patient against the antigen are selected bybinding to the antigen of interest, as described in Zebedee, et al.Proc. Natl. Acad. Sci. USA 89: 3175-3179 (1992), Burton et al., Proc.Natl. Acad. Sci. USA 88: 10134-10137 (1991), and Barbas et al., Proc.Natl. Acad. Sci. USA 89: 10164-20168 (1991).

In some embodiments, additional rounds of screening are performed toincrease the affinity of the originally isolated antibody. For example,in some embodiments, the affinity of the antibody is enhanced byaffinity maturation, in which hypervariable antibody regions are mutatedto produce a large number of combinations, and the correspondingantibody variants are screened via phage display to select antibodieshaving the desired affinity for the antigen. In further embodiments, thesmall protein epitope can undergo in vitro evolution, as described inmore detail below, to increase its binding affinity for the antibody.Advantageously, in some embodiments, for example those in whichantibodies are produced by “vaccinating” a patient, an analogous processis carried out by the host immune system (e.g., via clonal selection) toproduce high affinity antibodies specific for the antigen of interest.

In other embodiments, the immune effector comprises a non-antibodymolecule capable of stimulating an immune response in the subjectreceiving treatment. For example, the immune effector may comprise a C3convertase, as described, e.g., in U.S. Pat. No. 6,268,485 to Farriesand Harrison, which is herein incorporated by reference. C3 convertaseis an enzyme that catalyzes the proteolytic conversion of C3 proteininto C3 a and C3 b, which conversion comprises a critical step in thegeneration of the complement system response. C3 b binds to cellsurfaces near its site of generation, where it mediates phagocytosis andother destructive immune responses. In some embodiments, C3 convertaseis modified or derivatized to confer reduced susceptibility toinhibitors, resistance to proteolytic cleavage, enhanced affinity forcofactors, or other modifications that enhance the effectiveness of theenzyme in stimulating a complement-mediated immune response. In someembodiments, the immune effector comprises a C5 convertase, a mannosebinding protein (MBP), or another molecule that stimulatescomplement-mediated immunity.

Linking C3 convertase to the therapeutics of the instant inventionallows a complement-mediated immune response to be directed to cells,pathogens, pathogen-infected cells, and/or other cells targeted fortreatment by binding of the target epitope binding protein to variableepitopes on the surface of the targeted cells. A therapeutic with C3convertase as the immune effector is illustrated in FIG. 9 . In apreferred embodiment, peptide sequences encoding proteins that bind C3convertase (the immune effector binding protein) and a target epitope(the target epitope binding protein) are identified, isolated, andoptionally bred by in vitro evolution or other techniques to increasetheir affinity to their target ligands. A bifunctional fusion protein isthen constructed comprising the target epitope binding domain, and theconvertase binding peptide as the immune effector binding domain (Ebbd).The therapeutic is administered to a patient in need of treatment, uponwhich the target epitope-binding domain binds to the surface of amalignant cell or other target, and the C3 convertase produces acomplement-mediated immune response, which includes the production ofmembrane attack complexes that mediate the destruction of the targetedcell.

In some embodiments, the therapeutic comprises a target epitope-bindingdomain and a C3 convertase binding domain. Upon being administered, suchtherapeutics bind a target epitope and recruit endogenous C3 convertaseto initiate a complement-mediated immune response. Such an approach isillustrated in FIG. 10 , which shows a therapeutic in which the immuneeffector binds the C1 component of the complement system. Upon beingadministered to a patient in need of treatment, the therapeutic binds tothe target epitope and recruits endogenous C1 to the cell surface, whereit induces a complement-mediated immune response.

In other embodiments, the immune effector component of the therapeuticbinds to and inhibits C1 q, C3, C5, and/or other molecules involved ingenerating complement-mediated immune responses, such as the proteinsdescribed in U.S. Pat. No. 5,650,389. Upon being administered to apatient in need of treatment, such therapeutics bind to the targetepitope and inhibit the immune response that would otherwise be directedat or near the binding site. For example, in some preferred embodiments,a patient is the recipient of transplanted cells (e.g., stem cells, orcells comprising a transplanted organ), and the targeted therapeutic iscapable of recognizing transplanted cells, and inhibiting an immuneresponse against such cells. In further embodiments, targetedtherapeutics for the inhibition of transplant rejection comprise atarget-binding domain that targets one or more epitopes of immune cellsthat recognize transplanted cells, for example epitopes that bind MHCantigens on the surfaces of transplanted cells, and the therapeuticagent is an immune effector capable of stimulating an immune response inthe patient against the anti-transplant immune cells.

Advantageously, in several embodiments, the methods provided herein arerapid and cost-effective enough to allow routine creation ofindividualized therapeutics. Therapeutics according to severalembodiments can preferably be created, from reception of the biopsymaterial to the completion of the therapeutic, in a reduced period oftime and for less money than other methods.

A bifunctional fusion protein for use in the invention can be producedaccording to standard recombinant DNA techniques known to those skilledin the art (e.g., Sambrook, J. et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989)).In some embodiments, the purified bifunctional protein can be reactedwith the immune effector, yielding a conjugated effector-bifunctionalprotein complex, as shown FIG. 7 , which comprises the therapeutic. Thebifunctional protein can also be chemically bound to the immune effectorthrough means known in the art.

In some embodiments, a protein coupling agent, such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexaned-iamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethyl-enediamine), diisocyanates (such astolyene 2,6-diisocyanate), or bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene), is used to link two or more proteincomponents comprising the therapeutic.

In some embodiments, a recombinant fusion protein is prepared by:linking a first polynucleotide sequence encoding atarget-epitope-binding protein, or derivatives, fragments or subunitsthereof, to a second polynucleotide sequence encoding an epitope orother protein that binds the immune effector to generate a chimericcoding sequence; subcloning the chimeric coding sequence into anexpression vector; transfecting a cell with the expression vector; andpurifying the fusion protein expressed by the transfected cell. In someembodiments, the chimeric polynucleotide can include an initiationsequence appropriate for prokaryotic and/or eukaryotic in vitrotranslation systems, and/or a selectable marker.

In some embodiments, the bifunctional protein complex is made by invitro translation of an mRNA oligo that includes the mRNA encoding bothproteins and, in some embodiments, the major histocompatibility complexI and/or II. The cDNA for the mRNA sequences can be synthesized orobtained commercially, and transcribed by PCR to obtain sufficient mRNA,as shown in FIG. 3 . In various embodiments, the oligo is formed byligating mRNA encoding the immune effector-binding domain and the mRNAencoding the target epitope-binding domain. In some cases this fusioncan be connected through an mRNA bridge that codes for hydrophilic aminoacids, as illustrated in FIG. 6 . The mRNA can then be translated invitro using prokaryotic or eukaryotic translation systems, and theresultant bifunctional protein can purified by gel electrophoresis orany other method known in the art.

The present fusion proteins can also comprise one or more numerous othercomponents that enhance the utility of the chimeric proteins. Forexample, the proteins can be designed to contain an epitope tag useful,for example, to facilitate purification of the fusion proteins. Forexample, the chimeric coding sequence can be modified to encode two ormore neighboring histidine residues, for example, in the amino orcarboxy terminus of the peptide. Histidine residue insertion can bereadily accomplished by the splice-by-overlap extension methodology, byincorporating histidine-encoding CAT and CAC triplet codons into the PCRprimers at suitable locations in the coding sequence. Histidine-modifiedproteins can be efficiently and quantitatively isolated bynickel-sepharose chromatography methods known in the art.

In some embodiments, therapeutics provided herein may also be conjugatedto a second molecule, such as a therapeutic agent (e.g., a cytotoxicagent). For example, the therapeutic agent includes, but is not limitedto, an anti-tumor drug, a toxin, a radioactive agent, a cytokine, asecond antibody or an enzyme. Examples of cytotoxic agents include, butare not limited to ricin, ricin A-chain, doxorubicin, daunorubicin,taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D,diphteria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, arbrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,Sapaonaria Officinalis inhibitor, maytansinoids, and glucocorticoid andother chemotherapeutic agents, as well as radioisotopes such as ²¹²Bi,¹³¹I, ⁹⁰Y, and ¹⁸⁶Re. Suitable detectable markers include, but are notlimited to, a radioisotope, a fluorescent compound, a bioluminescentcompound, chemiluminescent compound, a metal chelator or an enzyme. Thetherapeutics of the invention may also be conjugated to an anti-cancerpro-drug activating enzyme capable of converting the pro-drug to itsactive form. See, for example, U.S. Pat. No. 4,975,287.

In some preferred embodiments, the secondary therapeutic agent has acomplementary mode of action with the primary therapeutic agent. Forexample, in some embodiments, the primary and secondary therapeuticagents act against different aspects of a signal transduction systeminvolved in the etiology of a cancer or other disease, leading toenhanced efficacy, fewer side effects, an improved therapeutic index,and/or other benefits relative to tailored therapeutics bearing theprimary and/or secondary therapeutic agents only. In some embodiments,the first therapeutic agent potentiates the second therapeutic agent, orvice versa, or the first and second therapeutic agents exhibit asynergistic enhancement in one or more aspects of treatment. Methods forassessing synergism, potentiation, and other combined pharmacologicaleffects are known in the art, and described, e.g., in Chou and Talalay,Adv Enzyme Regul., 22:27-55 (1984), incorporated herein by reference.

In some embodiments, the therapeutic agent is conjugated to an antibodyimmune effector component of the therapeutic. Techniques for conjugatingor joining therapeutic agents to antibodies are well known (see, e.g.,Arnon et al., “Monoclonal Antibodies For Immuno-targeting Of Drugs InCancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeldet al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,“Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.),Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, inMonoclonal Antibodies '84: Biological And Clinical Applications,Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982)).

Depending on the embodiment, the therapeutics produced according to themethods disclosed herein are suitable for administration by methods suchas intravenous injection, intramuscular injection, topicaladministration, oral ingestion, rectal administration, and inhalation.Alternatively, the therapeutics of the present invention can bedelivered directly to the site of the malignancy (or other target site).The therapeutic may be administered in admixture with a pharmaceuticallyacceptable carrier. Any such carrier can be used according to thepresent methods, as long as compatibility problems do not arise. Aneffective amount of the present recombinant fusion protein should beadministered to the patient. The term “effective amount” refers to thatamount of the fusion protein needed to bring about the desired response.

In some preferred embodiments, therapeutics made according to methodsproviding herein inhibit proliferation and/or induces apoptosis of cellsbearing the epitope against which the epitope binding protein wasscreened. For example, in some embodiments, the epitope-binding portionof the therapeutic complex can bind with the epitopes expressed on thesurface of malignant cells, or other targets. Once bound, theIR-eliciting antibody component of the complex stimulates the immunesystem to attack and eliminate the tagged cells, while sparing thenormal cells, as illustrated in FIG. 8 .

In various embodiments, the tailoring of individualized therapeuticsprovided herein for binding to patient- and/or disease-specific targetsis made possible by utilizing novel methods for linking proteins totheir corresponding mRNAs (as “cognate pairs”). In some preferredembodiments, protein libraries are prepared comprising a large number ofcognate pairs, and the libraries are screened for cognate pairs thatbind to a target of interest, such as the individualized targetsdescribed herein.

Various aspects of the present invention use modified tRNA and/or mRNAmolecules to link translated protein products to their correspondingmRNAs via a tRNA linker, forming a “cognate pair.” In severalembodiments, mRNAs having unknown sequences are expressed in an in vitrotranslation system, for example from an mRNA library, and theircorresponding proteins are screened for one or more desiredcharacteristics, such as binding to the target epitope-binding domain,or another ligand of interest, and/or selectivity over one or moreadditional ligands, such as epitopes displayed by healthy cells. Infurther embodiments, proteins and their linked nucleic acids identifiedin one or more rounds of selection are modified, for example throughnucleic acid evolution (FIG. 4 ), to produce proteins with enhancedaffinities for the target ligand. Proteins having desiredcharacteristics, such as a high affinity for the target ligand, can thenbe produced in large quantities using standard cloning techniques byisolating their corresponding mRNA from the protein-mRNA cognate pairs.

In some preferred embodiments, cognate pairs are formed using aeukaryotic in vitro translation system, such as rabbit reticulocytelysate (RLL), wheat germ, E. coli, or yeast lysate systems. However, itis understood by the skilled artisan that any in vitro translationsystem can be used, including in situ systems, as well as hybridsystems, which combine components of different systems. For example, insome embodiments, one or more prokaryotic factors are used in aeukaryotic translation system, such as translation suppressor proteins(see e.g., Geller and Rich Nature 283:41 (1980); Edwards et al PNAS88:1153 (1991); Hou and Schimmel Biochem 28:6800 (1989), all hereinincorporated by reference). In some embodiments, one or more tRNAs ortRNA analogs are charged in a prokaryotic system and then purifiedaccording to established methods (Lucas-Lenard and Haenni, PNAS 63:93(1969), herein incorporated by reference) for use in a eukaryoticsystem.

In various embodiments, proteins comprising cognate pairs are linked totRNA or a tRNA analog by the action of ribosomal peptidyl transferase.In some embodiments, proteins are linked to a stable aminoacyl tRNAanalog (SATA). In some embodiments, the SATA is a tRNA with an aminoacid or amino acid analog attached to its 3′ end via a stable bond,relative to the corresponding high-energy ester bond in the nativestructure. When the SATA recognizes a particular codon, for example viahydrogen bonding, and accepts a nascent peptide chain by the action ofthe ribosomal peptidyl transferase, the stable aminoacyl bond preventsthe detachment of the tRNA from the polypeptide by peptidyl transferase,and also preserves the tRNA-polypeptide structure during subsequentsteps.

In some embodiments, a SATA is created according to methods generallydescribed in Fraser and Rich, PNAS, 70:2671 (1973), herein incorporatedby reference, which involve the conversion of a tRNA, or tRNA analog, toa 3′-amino-3′-deoxy tRNA. This is accomplished by adding a3′-amino-3′-deoxy adenosine to the end of a native tRNA with tRNAnucleotidyl transferase after removing the native adenosine, and thencharging the modified tRNA with an amino acid with the respectiveaminoacyl tRNA synthetase (aaRS). In some embodiments, the aaRS chargesthe tRNA on the 3′, rather than the 2′, hydroxyl, linking the amino acidto the tRNA by a stable amide bond, rather than the usual labilehigh-energy ester bond. Thus, when the SATA accepts a peptide fromribosomal peptidyl transferase it will stably hold the peptide and beunable to donate it to another acceptor.

In certain embodiments, tRNAs aminoacylated via a 3′ amide bond may notcombine with the elongation factor EF-TU, which assists in binding tothe A site (e.g., Sprinzl and Cramer, Prog. Nuc. Acid Res. 22:1 (1979),herein incorporated by reference). Such modified tRNAs do, however, bindto the A site. This binding of 3′ modified tRNAs can be increased bychanging the Mg⁺⁺ concentration (Chinali et al., Biochem. 13:3001(1974), herein incorporated by reference). The appropriateconcentrations of and/or molar ratios of SATA and Mg²⁺ can be determinedempirically. For example, if the concentration or A site avidity of aSATA is too high, the SATA may compete with native tRNAs for non-cognatecodons, stalling translation. Alternatively, if the concentration or Asite avidity of SATA is too low, the SATA might fail to effectivelycompete with release factors, preventing it from stably accepting thenascent peptide.

While the elongation factor is also believed to aid in proofreadingcodon-anticodon recognition, the absence of this source of proofreadingwould not be expected to interfere with methods provided herein. Withoutbeing bound to a particular mechanism, it is believed that the errorrate in the absence of elongation factor and associated GTP hydrolysisis approximately 1 in 100 for codons one nucleotide away (Voet and Voet,Biochemistry 2^(nd) ed. pp. 1000-1002 (1995), John Wiley and Sons,herein incorporated by reference). In some preferred embodiments, UAA isused as the linking codon. UAA has 7 non stop codons that differ from itby one amino acid, which comprises 7/61, or about 11.5% of the non stopcodons. Thus, the probability of miscoding a given codon can beestimated as (0.01)(0.115)=1.15×10⁻³ miscodes per codon, or about onemiscode every 870 codons, a frequency that would not substantiallyimpair performance of various methods described herein. In someadditional embodiments, UAG can be used as the linking codon withoutsubstantial impairment due to the absence of elongation factor-mediatedproofreading.

In some embodiment, the SATA is a tRNA, or tRNA analog, with one or moremodified bases in the acceptor stem, or another region of the molecule.Various methods for producing tRNAs with acceptor stem modification areknown in the art, and are described, for example, in Sprinzl and Cramer,Prog. Nuc. Acid Res., 22:1 (1979), herein incorporated by reference. Insome embodiments, a tRNA is modified with a puromycin moiety, such thatthe tRNA mimics aminoacyl-Tyr tRNA and is incorporated into the nascentpolypeptide, terminating translation. In some embodiments, acceptorstem-modified tRNAs are formed from “transcriptional tRNA”, wherein thesequence of the tRNA itself, rather than post-transcriptionalprocessing, leads to the atypical and modified bases. TranscriptionaltRNAs are capable of functioning as tRNAs (see e.g., Dabrowski et al.,EMBO J. 14:4872, 1995; and Harrington et al., Biochem. 32: 7617, 1993,both herein incorporated by reference). Transcriptional tRNA can beproduced by methods known in the art, such as transcription, or byconnecting commercially available RNA sequences (e.g., from DharmaconResearch Inc., Boulder, Colo.) together, piece-wise as in FIG. X, or bysome combination of established methods. For example, with reference toFig. X, the 5′ phosphate and 3′ puromycin are commercially availableattached to oligoribonucleotides, which can be connected together usingT4 DNA ligase (e.g., Moore and Sharp, Science 256: 992, (1992), hereinincorporated by reference) or alternatively, T4 RNA ligase (Romaniuk andUhlenbeck, Methods in Enzymology 100:52 (1983), herein incorporated byreference).

Additional methods for producing modified tRNAs are known in the art,and are described, e.g., in Chinali et al., Biochem. 13:3001 (1974) andKrayevsky and Kukhanova, Prog. Nuc. Acid Res 23:1 (1979), both hereinincorporated by reference.

In some embodiments, the tRNA is a nonsense suppressor tRNA comprising amodified or unmodified tRNA, or tRNA analog, that recognizes a stopcodon or a pseudo-stop codon, preferably by codon-anticodon hydrogenbonding, such that translation is terminated when the nascent protein isattached to the tRNA by peptidyl transferase. In some embodiments, thenonsense suppressor tRNA has 3′ modifications and/or sequences thatconform to the Yarus extended anticodon rules (Yarus, Science218:646-652, 1982, herein incorporated by reference). A “pseudo stopcodon,” as defined herein, refers to a codon which, while not naturallya nonsense codon, prevents a message from being further translated. Apseudo stop codon can comprise a codon recognized by a “stable aminoacyltRNA analog,” or SATA, as described herein, or a codon for which tRNAbearing a complementary anticodon is substantially depleted or absent,such that translation is terminated when the absent tRNA is required,i.e. at the pseudo stop codon. One skilled in the art will appreciatethat are numerous ways to create a pseudo stop codon, as defined herein.

In some preferred embodiments, the tRNA is a native tRNA, linked to thenascent polypeptide via a native peptide bond. In some embodiments, theSATA is a tRNA that is unmodified at the 3′ end, but which may have oneor more modifications to the anticodon loop and/or other regions of themolecule. In various embodiments, the use of native tRNAs and/or tRNAsthat are unmodified at the 3′ end results in improvements in variousselection methods described herein, giving rise to quicker, lesserror-prone, more efficient, more cost-effective, and/or higher yieldmethods. While not being bound by a particular theory, it is believedthat, under certain conditions, puromycin (and similar linkers) canresult in lower yields due to interference with the interaction betweenelongation factor(s) and tRNAs.

In one embodiment of the invention, the crosslinker is an agent thatchemically or mechanically links two molecules together. In oneembodiment, the crosslinker is an agent that can be activated to formone or more covalent bonds with tRNA and/or mRNA. In one embodiment, thecrosslinker is a sulfur-substituted nucleotide. In another embodiment,the crosslinker is a halogen-substituted nucleotide. Examples ofcrosslinkers include, but are not limited to, 2-thiocytosine,2-thiouridine, 4-thiouridine, 5-iodocytosine, 5-iodouridine,5-bromouridine and 2-chloroadenosine, aryl azides, and modifications oranalogues thereof. In one embodiment, the crosslinker is psoralen or apsoralen analog.

In some preferred embodiments, a psoralen is monoadducted to a tRNA, ora tRNA analog (e.g., a 3′ modified SATA), for example by connecting apsoralen linked oligonucleotide (FIG. 3 ), or by monoadduction (FIG. 4), to a native or modified tRNA or tRNA analog, preferably to theanticodon or another site distinct from the linkage to the polypeptide.When irradiated with UV light of a desired wavelength, a covalentpsoralen crosslink is formed between the SATA and mRNA, as described inmore detail below. In some embodiments, the anticodon or other portionof a tRNA is derivatized with a non-psoralen moiety capable of forming acrosslink to the mRNA, such as 2-thiocytosine, 2-thiouridine,4-thiouridine 5-iodocytosine, 5-iodouridine, 5-bromouridine,2-chloroadenosine, aryl azides, and modifications or analogues thereof.These and other cross-linkers are known in the art, and are availablecommercially, for example from Ambion, Inc. (Austin, Tex.), Dharmacon,Inc. (Lafayette, Colo.), and other well-known manufacturers ofscientific materials.

In various embodiments, a SATA-polypeptide complex or a tRNA-polypeptidecomplex is linked to the mRNA encoding the polypeptide via a linkermoiety, which can be located on the mRNA and/or on the tRNA. In somepreferred embodiments, the mRNA comprises a cross-linker, preferably ator near a stop codon at the 3′-end of the transcript, and the mRNA-tRNAlinkage is mediated entirely by the mRNA-based cross-linker. In furtherembodiments, the tRNA is unmodified at its 3′-end (e.g., a nonsensesuppressor tRNA). In some preferred embodiments, an mRNA comprises apseudo-stop codon located at the end of the translatable reading frame.A pseudo-stop codon can be effectively placed at the end of a readingframe where the pseudo-stop codon is located at the 3′ end of the mRNA,where the translation system is depleted of tRNAs corresponding tocodons 3′ to the pseudo-stop codon, and/or where 3′-modified tRNAscorresponding to the pseudo-stop codon are used, rendering thetranscript untranslatable and incapable of activating release factors.In some embodiments, the mRNA comprises a stop codon corresponding to atRNA stop anticodon. Advantageously, a stop codon/anticodon pair selectsfor full-length transcripts. One skilled in the art will understand thatan mRNA not having a stop codon may also be used and that any codon ornucleic acid triplet may be used.

In some methods, a SATA is attached to the translated message by apsoralen cross link between the codon and anticodon. Psoralen crosslinks are preferentially made between sequences that containcomplementary 5′ pyrimidine-purine 3′ sequences, especially UA or TAsequences (Cimino et al., Ann. Rev. Biochem. 54:1151 (1985), hereinincorporated by reference). The codon coding for the SATA, or thelinking codon, can be PYR-PUR-X or X-PYR-PUR, so that several codons maybe used for the linking codon. Conveniently, the stop or nonsense codonshave this configuration. Using a codon that codes for an amino acid mayrequire minor adjustments to the genetic code, which could complicatesome applications. Therefore, in a preferred embodiment, a stop codon isused as the linking codon and the SATA functions as a nonsensesuppressor in that it recognizes the linking codon. One skilled in theart, however, will appreciate that, with appropriate adjustments to thesystem, any codon can be used.

In some preferred embodiments, the SATA or peptidyl-tRNA is cross-linkedto the translated mRNA, for example between the codon and anticodon, bya psoralen cross-link, or by a cross-link formed from the groupconsisting of: 2-thio cytosine, 2-thio uridine, 4thio uridine5-iodocytosine, 5-iodouridine, 5-bromouridine, 2-chloroadenosine, andaryl azides. Psoralen cross links are, in some embodiments,preferentially made between sequences that contain complementary 5′pyrimidine-purine 3′ sequences, especially UA or TA sequences (Cimino etal., Ann. Rev. Biochem. 54:1151 (1985), herein incorporated byreference). In some embodiments, non-psoralen crosslinkers or arylazides are used and in certain embodiments, are particularlyadvantageous because they are less stringent in their requirements andtherefore increase the possible codon-anticodon pairs.

In various embodiments, translation terminates when the nascent proteinis attached to a SATA by the peptidyl transferase and/or when the end ofthe reading frame is reached. When a large number of ribosomes are inthis position, the SATA and the mRNA are cross-linked by application ofUV light. In a preferred method, cross-linking is accomplished byforming a psoralen crosslink upon irradiation with UV light, preferablyin the range of 320 nm to 400 nm. Psoralens comprise a furan side and apyrone side, and they readily intercalate between complementary basepairs in double stranded DNA, RNA, and DNA-RNA hybrids (Cimino et al.,Ann. Rev. Biochem. 54:1151 (1985), herein incorporated by reference). Insome preferred embodiments, psoralen cross-linking forms monoadducts,described more fully below, that are either pyrone sided or furan sidedmonoadducts. Upon further irradiation, the furan sided monoadducts canbe covalently crosslinked to complementary base pairs, whereas thepyrone sided monoadducts cannot be further crosslinked. The formation offuran-sided psoralen monoadducts (MM) is achieved according toestablished methods. In additional embodiments, psoralen can also beattached at the end of the reading frame of the message.

Methods for large scale production of purified MAf on oligonucleotidesare described in the literature (e.g., Speilmann et al., PNAS 89:4514,1992, herein incorporated by reference), as are methods that requireless resources, but have some non-cross-linkable pyrone sided psoralenmonoadduct contamination (e.g., U.S. Pat. No. 4,599,303; Gamper et al.,J. Mol. Biol. 197:349 (1987); Gamper et al., Photochem. Photobiol. 40:29(1984), both herein incorporated by reference). In several embodimentsof the current invention, psoralen labeling is accomplished by usingeither method. In a preferred embodiment, furan sided monoadducts willbe created using visible light, preferably in the range of approximately400 nm-420 nm, according to the methods described in U.S. Pat. No.5,462,733 and Gasparro et al., Photochem. Photobiol. 57:1007 (1993),both herein incorporated by reference. In one aspect of this invention,a SATA with a furan sided monoadduct or monoadducted oligonucleotidesfor placement on the 3′ end of mRNAs, along with a nonadducted SATA areprovided as the basis of a kit.

In one embodiment, the formation and reversal of monoadducts andcrosslinks are performed according to the methods of Bachellerie et al.(Nuc Acids Res 9:2207 (1981)), herein incorporated by reference. In apreferred embodiment, efficient production of monoadducts, resulting inhigh yield of the end-product, is accomplished using the methods ofKobertz and Essigmann, J. A. Chem. Soc. 1997, 119, 5960-5961 and Kobertzand Essigmann, J. Org. Chem. 1997, 62, 2630-2632, both hereinincorporated by reference.

Other methods for connecting the mRNA to its protein can be used, aswell as methods of phage display.

In several embodiments, appropriate concentrations of SATA and Mg⁺⁺ areused in the in vitro translation system in the presence of the mRNAmolecules, causing translation to cease when ribosomes reach a stop, orpseudo-stop, codon which permits the SATA to accept the peptide chain,as described above. After a short time, a substantial proportion and/ornumber of the stop or pseudo-stop codons are occupied by SATAs withinribosomes, and in some embodiments, the system is then irradiated withUV light to generate cross-links between the tRNA-polypeptides and theircorresponding mRNAs. In several embodiments, the ribosomes are releasedor denatured after cross-linking of to mRNA, preferably by the depletionof Mg⁺⁺ through dialysis, dilution, or chelation. One skilled in the artwill understand that other methods, including but not limited to,denaturation by changing the ionic strength, the pH, or the solventsystem can also be used to release cognate pairs from associatedribosomes and/or other translation factors.

In various embodiments, cognate pairs are selected based on one or moredesired characteristics. In some embodiments, the selection of cognatepairs is based upon the binding of a target cell, protein, and/or otherbiomolecule, as determined by any of a variety of established methods,including, but not limited to, arrays, affinity columns,immunoprecipitation, and the like. In some preferred embodiments,selection criteria are measured using a high throughput screeningprocedure. The selection can be positive or negative in variousembodiments, according to the desired characteristics of the therapeuticagent. In several preferred embodiments, mRNAs whose sequences areunknown are expressed (e.g., in the form of an mRNA library from apatient, tissue, or other source of interest) and linked to theirencoded polypeptides using methods described herein, and the cognatepairs are screened to select for desired properties. For example, insome preferred embodiments, cognate pairs are assayed for binding to aligand of interest, such as an Fab idiotype displayed by a malignantcell, or other surface characteristic of a target cell. mRNA can beisolated for proteins exhibiting the desired binding characteristics,and large quantities of the protein can be produced using standardmolecular cloning techniques known in the art. As described in moredetail herein, the protein of interest can then be incorporated into amodular therapeutic agent, for example to target the agent to a patient-and/or disease-specific target.

In various embodiments, the selected cognate pairs can be those that dobind well to a ligand or those that do not. For instance, for a proteinto accelerate a thermodynamically favorable reaction, i.e., act as anenzyme for that reaction, it should bind both the substrate and atransition state analog. However, the transition state analog should bebound much more tightly than the substrate.

This is described by the equation

$\frac{k_{enzyme}}{k_{\varphi\;{enzyme}}} = \frac{K_{trans}}{K_{subst}}$

where the ratio of the rate of the reaction with the enzyme, k_(enzyme)to the rate without, k_(enzyme), is equal to the ratio of the binding ofthe transition state to the enzyme K_(trans) over the binding of thesubstrate to the enzyme K_(subst) (Voet and Voet, Biochemistry 2^(nd)ed. p. 380, (1995), John Wiley.

In some preferred embodiments, proteins which compete poorly for bindingto the substrate but compete well for binding to the transition stateanalog are selected. Operationally, this may be accomplished by takingthe proteins that are easily eluted from a matrix with substrate orsubstrate analog bound to it and are the most difficult to remove frommatrix with transition state analog bound to it. By sequentiallyrepeating this selection and reproducing the proteins throughreplication and translation of the nucleic acid of the cognate pairs, animproved enzyme should evolve. Affinity to one entity and lack ofaffinity to another in the same selection process is used in severalembodiments of the current invention. In some additional embodiments,cognate pairs can be selected according to one or more properties of themRNA portion.

There are many methods known in the art for identifying epitopesexpressed by normal and malignant cells. For example, in someembodiments, a peptide microarray can be used to isolate cell-specificmarker peptides from a combinatorial library, as described, e.g., byAina et al, “Therapeutic Cancer Targeting Peptides,” Biopolymers66:184-199 (2002). In some preferred embodiments, the protein-mRNAcomplex library is reacted an isolated population of malignant cells,and the degree of binding to the malignant cell epitope is measuredrelative to binding observed against a population of normal cells. Insome embodiments, proteins are selected having substantial affinity formalignant cells, with substantially lower or no affinity for normalcells. For example, in some embodiments, polypeptides are identifiedthat bind an epitope or other target of interest with an affinity ofless than about 10 μM, preferably less than about 1 μM, more preferablyless than about 0.1 μM, and even more preferably less than about 10 nM.In some preferred embodiments, a polypeptide has an affinity for theepitope or other target of less than about 1 nM. Screening methods canbe carried out with the potential ligands (e.g., proteins linked totheir cognate mRNA) and targets (e.g., cells targeted for treatment) ina variety of orientations. For example, in some embodiments, malignantcells are presented on a planar surface, such as a glass slide, and areexposed to a solution containing mRNA-protein cognate pairs. Boundproteins (cognate pairs) can be detected via a variety of methods knownin the art. For example, in some embodiments, the cognate pairs arederivatized, preferably on the mRNA and/or the tRNA linker, with adetectable probe, such as a biotin moiety, which can then be detectedwith a secondary reporter probe, for example using avidin coatedmagnetic beads. The resultantidiotype-(protein:mRNA)-biotin-avidin-magnetic bead complex can beidentified with a Ventana 320 automated immunohistochemistry system(Ventana Medical Systems, Tucson, Ariz.), or a similar system, asdescribed, for example, in Davis et al., Clinical Cancer Research 5:611-615, (1999).

The method can further comprise providing a plurality of distinctnucleic acid-polypeptide complexes, providing a ligand with a desiredbinding characteristic, contacting the complexes with the ligand,removing unbound complexes, and recovering complexes bound to theligand.

Several methods of the current invention involve the evolution ofnucleic acid molecules and/or proteins. In some embodiments, suchmethods comprise amplifying the nucleic acid component (as RNA, orcorresponding cDNA) of the recovered complexes and introducing variationto the sequence of the nucleic acids, for example by error-prone PCR, asdescribed, e.g., in Cadwell et al., PCR Methods Appl., 2: 28 (1992),incorporated herein by reference, in vitro recombination, described,e.g., in U.S. Pat. No. 5,605,793, mutagenesis, described, e.g., in U.S.Pat. No. 5,830,721, “DNA shuffling,” described, e.g., in Coco et al.,Nat Biotechnol, 19(4):354-9 (2001), and/or other methods known in theart. In some preferred embodiments, at least one amino acid substitutionis introduced at each position in the protein. In further embodiments,the method further comprises translating polypeptides from the amplifiedand varied nucleic acids, linking them together using tRNA, andcontacting them with the ligand to select another new population ofbound complexes. Several embodiments of the present invention useselected protein-mRNA complexes in a process of in vitro evolution,especially the iterative process in which the selected mRNA isreproduced with variation, translated and again connected to cognateprotein for selection.

The Replication Threshold

A nominal minimum number of replications for efficient evolution may beestimated using the following formulae. If there is a sequence which isn sequences in length, with a selective improvement r mutations awaywith a mutation rate of p, the probability of generating the selectiveimprovement on replication may be determined as follows:

For r=1, probability of a mutation at the right point, p, times theprobability that it mutated to the right one of the three nucleotidesthat are different from the starting point, ⅓, times the probabilitythat the other n-1 sites remain unmutated, (1-p)^((n−r)), or

$P_{r} = {\left( \frac{p}{3} \right)^{1}\left( {1 - p} \right)^{({n - 1})}}$

where, P=the probability of attaining a given change r mutations away.More generally, for all r values:

$P_{r} = {\left( \frac{p}{3} \right)^{r}\left( {1 - p} \right)^{({n - r})}}$

It is instructive to compare the chances of finding an advantage onemutation away with the chances three mutations away. This is because,given the triplet genetic code, any given codon can only change intonine other codons in one mutation. Indeed, it turns out that no codoncan actually change into nine other amino acid codes in one mutation.The maximum number of amino acids that can be accessed in one mutationis seven amino acids and there are only eight codons of the sixty-fourthat can do this. Most codons have five or six out of nineteen otheramino acids within one mutation. To reach all nineteen amino acids thatare different from the starting one requires, in general, threemutations. These three mutations cannot be sequential since the twointervening ones will not, in general, be selectively advantageous.Therefore we need to use steps that are, at least, three mutations insize (r=3) to use all 20 amino acids.

For a mutation rate of 0.0067, which is that reported for “error-pronePCR”, using a message of 300 nucleotides, which gives a short protein of100 amino acids:P ₃=1.51×10⁻⁹

Therefore, one would expect to need a threshold of:

$\frac{1}{1.51 \times 10^{- 9}} = {6.64 \times 10^{8}}$

replications at that mutation rate to reasonably expect to reach thenext amino acid that is advantageous. This is not the replication to usesince the binomial expansion shows that over ⅓ of trials (actually about1/e) would not contain the given sequence with selective advantage.

A poisson approximation for large n and small p for a given μ can becalculated so that we can compute the general term when n is, say, ofthe order 10⁹ and p is of the order 10⁻⁹. The general term of theapproximation is:

$\frac{\mu^{r}}{{r!}e^{\mu}}$

An amplification factor of greater than approximately 6/P ensures thatevolution will progress with the use of all amino acids. This is usefulwhen the production of novel proteins precludes the use of “shuffling”of preexisting proteins.

Limits on Purification

Given a reversible binding where B and C compete for A:

$\begin{matrix}\left. {AB}\leftrightarrow{A + B} \right. & \; \\{\left. {A\; C}\leftrightarrow{A + C} \right.{k_{B} = \frac{\lbrack A\rbrack\lbrack B\rbrack}{\lbrack{AB}\rbrack}}{k_{C} = \frac{\lbrack A\rbrack\lbrack C\rbrack}{\left\lbrack {A\; C} \right\rbrack}}} & \; \\{\lbrack B\rbrack = {k_{B}\frac{\lbrack{AB}\rbrack}{\lbrack A\rbrack}}} & (1) \\{\lbrack C\rbrack = {k_{C}\frac{\left\lbrack {A\; C} \right\rbrack}{\lbrack A\rbrack}}} & (2)\end{matrix}$

The total concentrations can be expressed as follows:[B]_(T)=[B]+[AB]  (3)[C]_(T)=[C]+[AC]  (4)

Dividing (3) by (4):

$\frac{\lbrack B\rbrack_{T} = {\lbrack B\rbrack + \lbrack{AB}\rbrack}}{\lbrack C\rbrack_{T} = {\lbrack C\rbrack + \left\lbrack {A\; C} \right\rbrack}}$

And substituting (1) and (2) for [B] and [C]:

$\frac{\lbrack B\rbrack_{T} = {{k_{B}\left\lbrack \frac{AB}{A} \right\rbrack} + \lbrack{AB}\rbrack}}{\lbrack C\rbrack_{T} = {{k_{C}\left\lbrack \frac{A\; C}{A} \right\rbrack} + \left\lbrack {A\; C} \right\rbrack}}$

Rearranging the equation gives the following results:

$\frac{\lbrack B\rbrack_{T}}{\lbrack C\rbrack_{T}} = \frac{\lbrack{AB}\rbrack\left( \frac{k_{B} + \lbrack A\rbrack}{\lbrack A\rbrack} \right)}{\left\lbrack {A\; C} \right\rbrack\left( \frac{k_{C} + \lbrack A\rbrack}{\lbrack A\rbrack} \right)}$

Canceling the [A]'s in the numerator and denominator:

$\frac{\lbrack B\rbrack_{T}}{\lbrack C\rbrack_{T}} = \frac{\lbrack{AB}\rbrack\left( {k_{B} + \lbrack A\rbrack} \right)}{\left\lbrack {A\; C} \right\rbrack\left( {k_{c} + \lbrack A\rbrack} \right)}$

Finally, rearranging the equation provides the following equation:

$\frac{\lbrack{AB}\rbrack}{\left\lbrack {A\; C} \right\rbrack} = \frac{\lbrack B\rbrack_{T}\left( {k_{C} + \lbrack A\rbrack} \right)}{\lbrack C\rbrack_{T}\left( {k_{B} + \lbrack A\rbrack} \right)}$$\frac{\left( {k_{C} + \lbrack A\rbrack} \right)}{\left( {k_{B} + \lbrack A\rbrack} \right)}\mspace{14mu}\left( {{Enrichment}\mspace{14mu}{Factor}} \right)$

The above factor is termed the “Enrichment Factor”. The ratio of thetotal components is multiplied by this factor to calculate the ratio ofthe bound components, or the enrichment of B over C. The maximumenrichment factor is k_(C)/k_(B), when the [A] is significantly smallerthan k_(C) or k_(B). When [A] is significantly greater than k_(C) ork_(B), the enrichment is 1, that is, there is no enrichment of one overthe other.

The enrichment is limited by the ratio of binding constants. To enrich ascarce protein that is bound 100 times as strongly as its competitors,the ratio of that protein to its competitors is increased by 1 millionwith 3 enrichments. To enrich a protein that only binds twice asstrongly as its competitors, 10 enrichment cycles would gain only anenrichment of ˜1000.

By an exactly analogous method an enrichment factor of selectingproteins that bind least well can be shown:

In the equation:

$\frac{\lbrack C\rbrack}{\lbrack B\rbrack} = \frac{{k_{C}\lbrack C\rbrack}_{T}\left( {\lbrack A\rbrack + k_{B}} \right)}{{k_{B}\lbrack B\rbrack}_{T}\left( {\lbrack A\rbrack + k_{C}} \right)}$

The enrichment here is maximal at [A]>k_(A) or k_(B).

$\frac{k_{C}\left( {\lbrack A\rbrack + k_{B}} \right)}{k_{B}\left( {\lbrack A\rbrack + k_{C}} \right)}$

Therapeutics Directed to Soluble Targets

As discussed above, the terms “soluble target” and “soluble agent” shallbe given their ordinary meaning and shall also refer to toxins, venoms,factors that have the capacity to alter biochemical pathways,biochemical agents, and the like that are not solid or tissue-based(e.g., they are present in the blood circulation of a subject as opposedto being a in or on a cell or mass of cells, etc.). Non-limitingexamples of soluble targets are shown in Tables 1-8, below.

TABLE 1 Animal or Insect Toxins Targeting Blood and Blood VesselsGeneral Target Toxin Producers Example Organism Examples of ToxinsProduced Blood Vessel Dilators Cephalopods octopus eledoisintachykinin-like peptides Dipteran insects mosquito sialokinins Frogstoad tachykinin-like peptides Hymenopteran bee tachykinin-like peptidesinsects Short-tailed shrews shrew blarinatoxin kallikrein Toxicoferanreptiles lizard peptidase S1 toxins Anticlotting Toxins Dipteran insectsmosquito aegyptin anopheline antiplatelet protein apyrase thrombostasinsanophelins Assassin bugs kissing bug pallidipin and related lipocalins(reduviidae) infestin and related kazal-type proteins rhodnius prolixusaggregation inhibitor-1 Leeches leech leech antiplatelet protein saratinhirudins ornatins Snakes rattlesnake snake venom metalloproteinasesC-type lectin toxins factor Xa-like proteases bothrojaracin Ticks tickmoubatin and related lipocalins apyrase savignin and related kunitz-typeproteins variegin variabilin Fleas flea apyrase Hymenopteran bee apyraseinsects Caterpillars wooly bear lopap lonomin III

TABLE 2 Animal or Insect Toxins Targeting Nervous and Muscle TissueGeneral Example Target Toxin Producers Organism Examples of ToxinsProduced Calcium Assassin bugs (reduviidae) kissing bug assassin bugtoxins Channel Blockers Cone snails cone snail ω-conotoxins Lampreyslamprey lamprey salivary CRISP Snakes rattlesnake calcicludine Spidersblack widow ω-neurotoxins Toxicoferan reptiles lizard CRISP toxinsPotassium- Cone snails cone snail K-conotoxin Channel BlockersHymenopteran insects bee apamin Scorpions scorpion short scorpion toxinsSea anemones sea anemone cnidaria kunitz-type proteinase inhibitors seaanemone type 3 potassium channel toxins Snakes rattlesnake dendrotoxinsSpiders black widow K-atracotoxins Toxicoferan reptiles lizard CRISPtoxins Sodium- Cone snails cone snail μ-O-conotoxins (pre- andpost-synaptic) Channel Blockers Spiders black widow hainantoxinsprotoxin-II huwentoxin-IV Sodium- Scorpions scorpion β-toxins ChannelActivators Spiders black widow μ-neurotoxins Sodium- Cone snails conesnail δ-conotoxins Channel Prolongers Irukandji jellyfish jellyfishirukandji-toxins Scorpions scorpion α-toxins Sea anemones sea anemonesea anemone sodium channel inhibitory toxins Spiders black widowδ-atracotoxins Nicotinic Cone snails cone snail α-conotoxins ReceptorAntagonists Snakes rattlesnake α-neurotoxins Muscarinic Scorpionsscorpion uncharacterized toxin or toxins Receptor Antagonists Snakesrattlesnake three-finger toxins phospholipase A2 toxins

TABLE 3 Plant Toxins Toxin Producers Toxin Produced Symptoms Induced byToxin Rosary pea abin inhibits protein synthesis Bitter almonds (prunusdulcis) amygdolin β-glucosidosis in gut releases cyanide Shikima plantanisatin respiratory paralysis Nux-vomica tree brucine stryhnine-liketoxin Water hemlock cicutoxin CNS toxin Delphinium delphinine cardiacactivity toxin Djenkon beans djenkolic acid kidney toxin Americanmayapple epipodophyllotoxin cytotoxin Gensing falcarinol contactdermatitis Ivy falcarinol contact dermatitis Gelonium plant geloninprotein synthesis toxin Cotton plant gossypol dehydrogenase enzymeinhibitor Cassava leaves and roots linamarin gut releases cyanide Lotuslotaustralin gut releases cyanide Lima beans lotaustralin gut releasescyanide Roseroot lotaustralin gut releases cyanide White cloverlotaustralin gut releases cyanide Cycad seeds β-methylamino-L-alanineneurotoxin Hemlock water dropwart oemanthotoxin CNS toxin Oleandersoleandrin neuro- and cardio-toxins Boraginaceae pyrrolizidane alkaloidsliver toxins Compositae pyrrolizidane alkaloids liver toxins Orchidaceaepyrrolizidane alkaloids liver toxins Leguminosiae pyrrolizidanealkaloids liver toxins Euphorbia (resin spurge latex) resiniferatoxinpain inducer Castor bean ricin severe allergin Foxglove saponin(digoxine) cardiac toxin Nightshade datura scopolamine neurotoxinNux-vomica tree (seeds) strychnine neurotoxin Locoweed swainsonineneurotoxin Abyssinian kale thionins cytotoxin Tutu plant tututoxinconvolsant

TABLE 4 Algae Derived Toxins Toxin Symptoms General Target ToxinProducers Produced Induced by Toxin Nervous Tissue Dinoflagellatessaxitoxin paralytic poisoning (in shellfish) Dinoflagellates domoic acidamnesic shellfish poisoning (in mussels, oysters, fish) Dinoflagellatesbrevetoxin neuro toxin (in shellfish) Dinoflagellates okadaic aciddiarrhetic poisoning (in shellfish) Hepatic Tissue Blue green algaemicrocystins liver failure

TABLE 5 Fungi Derived Toxins Toxin Producers Toxin Produced SymptomsInduced by Toxin Amanita α-amanitin deadly liver damage within 1-3 daysAmanitas phallotoxin gastrointestinal upset Cortinarius orellaninedeadly kidney failure within 3 weeks Omphalotus muscarine respiratoryfailure, sometimes deadly Gyromitra gyromitrin deadly neurotoxicity, GIupset, destruction of blood cells Coprinus coprine causes illness whenconsumed with alcohol A. muscaria ibotenic acid, muscimol hallucinogenicA. pantherina ibotenic acid, muscimol hallucinogenic A. gemmata ibotenicacid, muscimol hallucinogenic Psilocybe psilocybin, psilocinhallucinogenic Pleurotus ostreatus arabitol gastrointestinal irritationBoletus satanas bolesatine hemotoxin Claviceps purpurea ergotamineaffects vascular system, loss of limbs, death

TABLE 6 Bacterial Derived Toxins Toxin Producers Toxin Produced SymptomsInduced by Toxin Bacillus anthracis Anthrax toxin (EF) edema anddecreased phagocytic response Bordetella pertussis Adenylate cyclasetoxin (pertussis formation of ion-permeable pores in cell AC) membranesStaphylococcus aureus Alpha toxin formation of ion channel in cellplasma membrane Vibrio cholerae Cholera enterotoxin (Ctx) secretion ofwater, electrolytes leading to diarrhea Escherichia coli E. coli LTtoxin secretion of water, electrolytes leading to diarrhea Escherichiacoli E. coli ST toxins secretion of water, electrolytes leading todiarrhea Shigella dysenteriae E. coli Shiga toxin diarrhea, hemorrhagiccolitis, hemolytic O157:H7 uremic syndrome Clostridium perfringensPerfringens enterotoxin diarrhea Clostridium difficile ToxinA/ToxinBcell necrosis, bloody diarrhea, colitis Clostridium botulinum Botulinumtoxin flaccid paralysis Clostridium tetani Tetanus toxin spasticparalysis Corynebacterium Diphtheria toxin (Dtx) inhibits proteinsynthesis in target cells diphtheriae Pseudomonas aeruginosa Exotoxin Ainhibits protein synthesis Bacillus anthracis Anthrax toxin (LF)cytotoxicity of cells Bordetella pertussis Pertussis toxin (Ptx)interferes with metabolic regulation in cells Staphylococcus aureusExfoliatin toxin intraepidermal separation Staphylococcus aureusStaphylococcus enterotoxins causes massive activation of immune systemStaphylococcus aureus Toxic shock syndrome toxin (TSST- inflammation,fever, shock (acts on 1) vascular syst.) Staphylococcus pyogenesErythrogenic toxin inflammation, fever, shock

TABLE 7 Biowarfare Agents Agent Target Example Agent Symptoms Induced byAgent Nerve Agents Cyclosarin (“GF”) weakness, nausea; long-term neurofunctioning damage Sarin (“GB”) weakness, nausea; long-term neurofunctioning damage Soman (“GD”) vision, breathing difficulties;diarrhea; fatal in large doses Tabun (“GA”) vision, breathingdifficulties; diarrhea; fatal in large doses VX vision, breathingdifficulties; diarrhea; fatal in large doses VR breathing difficulties;slow heartbeat; paralysis in large doses VM vision, breathingdifficulties; diarrhea; fatal in large doses VG vision, breathingdifficulties; diarrhea; fatal in large doses VE vision, breathingdifficulties; diarrhea; fatal in large doses Insecticides (some)Novichok agents vision, breathing difficulties; diarrhea; fatal in largedoses Blood Agents Cyanogen chloride respiratory problems; convulsions;fatal in large doses Hydrogen cyanide respiratory problems; convulsions;fatal in large doses Arsines (most) weakness, fatigue, nausea, paralysisor death in large doses Vesicant Agents Sulfur mustard skin & eyeirritant, difficulty breathing; abdominal pain Nitrogen mustard skin &eye irritant, difficulty breathing; abdominal pain; seizures Lewisiteeye, respiratory irritant; diarrhea; seizures; low blood pressurePhosgene oxime skin hives; severe lung and eye irritant Pulmonary AgentsChlorine burning of eyes, nose, throat; nausea; skin blisters; pulmonaryedema Hydrogen chloride corrosive burns in eyes, nose, throat, upperrespiratory tract Nitrogen oxides eye, skin, respiratory irritation;pulmonary edema Phosgene burning of eyes, throat; shortness of breath;nausea Lachrymatory Agents Tear gas eye, nose, mouth, lung irritant;skin burns; nausea Pepper spray eye, nose, skin, respiratory irritant;neurogenic inflammation Incapacitating Agents Agent 15 confusion,hallucinations, blurred vision, rapid heart rate Quinuclidinyl benzilateconfusion, hallucinations, blurred vision, rapid heart rate CytotoxicProtein Agents Ricin respiratory distress, fever, nausea, vomiting,diarrhea, organ failure Abrin respiratory distress, fever, nausea,vomiting, diarrhea, organ failure

TABLE 8 Biopathway Modulators Factor Biological Action InterleukinsTyrosine kinase inducers Vascular Endothelial Growth Factors Cancer andother disorders (VEGF, A, B, C, D, E, F) Placental Growth Factor Cancerangiogenesis/vasculogenesis (PLGF) Tumor Necrosis Factor AlphaInflammation, arthritis Tumor Necrosis Factor Beta Kills infected cells,product of CD8⁺ T cells CC Chemokine Cell migration inducer(β-Chemokine) CC Chemokine ligands Migration inducer for monocytes, NKand dendritic cells (CCL-1, 15, 23, 28) Monocyte Chemo attractantProtein 1 (MCP- Inducer of monocytes to become tissue macrophages 1 orCCL2) RANTES (CCL-5) Attract CCR6 expressing T cells, eosinophils andbasophils Glutamic Acid-leucine-arginine Inducer of migration ofneutrophils (ELR) CXCL13 Chemo attractant for lymphocytes LymphotactinAlpha Attract T cell precursors to thymus (XCL-1) Lymphotactin BetaAttract T cell precursors to thymus (XCL-2) Fractalkine Adhesionmolecule (CX₃CL1) Interferon Type I, II, & III Anti viral and cancer,activate Nk cells and macrophages

It shall be appreciated, based on the non-limiting examples of solubleagents presented herein that the variety of soluble agents and thepotential deleterious effects that they induce is quite broad. However,in several embodiments the methods and therapies disclosed herein allowthe rapid, cost-effective, and specific generation and use ofbifunctional therapeutics that reduce, eliminate or otherwise diminishthe deleterious effects of exposure to such soluble agents.

In several embodiments, the generation of a bifunctional targetedtherapeutic is performed based on the exploitation of a known antigenand a sequence of mRNA that encodes that antigen (see e.g., FIGS.17A-17D). In several embodiments, a more general immune-mediatedapproach is used to generate the therapeutics (see e.g., FIGS. 20A-20D).However, in both such approaches, a specific protein that interacts withthe soluble agent is identified. Thus, the bifunctional (one portioninteracts with the soluble target while another interacts with one ormore components of the immune system) protein functions to snare thesoluble target and simultaneously (or subsequently) clear the solubleagent from a subject via an immune-mediated response.

In several embodiments, the bifunctional targeted therapeutics areparticularly advantageous because no therapy or treatment means forclearing the agent presently exists. In some embodiments, theselectivity the bifunctional targeted therapeutics enhances the efficacyof treatment relative to non-tailored therapeutics, due, for example, tothe non-selective activity of non-tailored therapeutics. In someembodiments, the therapeutics provided are more efficient at interactingwith a wider variety of soluble agents as the protein-target interactionpresents a wider scope of interactions that can be exploited. Forexample, use of an antibody based approach against certain solubleagents may not be particularly effective against agents having a lowimmunogenicity. In contrast, the use of targeted therapeutics providefor herein can, in some embodiments, exploit protein-proteininteractions between the soluble agent and the protein therapeutic(e.g., a steric relationship that is not highly immunogenic). As such,the possible ways of capturing or interacting with the soluble agent maybe greater, and in some embodiments, more effective, than simply relyingon antibody-based interactions. In additional embodiments, proteins thatinteract with soluble chemical agents are used in the generation of thebifunctional protein.

Generation of Bifunctional Targeted Therapeutics Therapeutics Exploitinga Known Antigen

In various embodiments, the generation of bifunctional therapeuticsprovided herein for binding to a variety of soluble agents is madepossible by utilizing novel methods for linking proteins to theircorresponding mRNAs (as “cognate pairs”). In some preferred embodiments,protein libraries are prepared comprising a large number of cognatepairs, and the libraries are screened for cognate pairs that bind to atarget of interest, such as the individualized targets described herein.Further information regarding the generation and use of a library ofproteins linked to their cognate mRNAs can be found in U.S. Pat. Nos.6,962,781; 7,351,812; 7,410,761; and 7,488,600 and the following U.S.patent application Ser. No. 11/813,849, filed May 2, 2008 (currentlypending) and Ser. No. 12/525,437, filed Jul. 31, 2009 (currentlypending). Each of the aforementioned Patents and patent applications areexpressly incorporated in their entirety by reference herein. As showngenerally in FIGS. 17A-17D, several embodiments, bifunctional targetedtherapeutics are produced that will be used in conjunction with a knownantigen. In FIG. 17A, a soluble target is shown on a solid substratethat is panned with protein-SATA-mRNA library under neutral and thenacid conditions, stimulating-plasma and endosome conditions. As shown inFIG. 17A, a soluble target of interest is immobilized on a solidsubstrate and panned with a library of proteins that are linked to theircognate mRNA (e.g., the mRNA sequences that code for that particularprotein). Entire proteins need not be used, fragments, or portions ofproteins are sufficient to interact with a soluble target in someembodiments. In several embodiments, the library is panned against thesoluble target under conditions similar to those in the bloodstream(e.g., about a neutral pH). In several embodiments, the library is alsopanned against the soluble target under acidic conditions, simulatingthe endosome or other cellular complex in which the degradation of thesoluble target may occur. Based on the panning of the library aparticular protein (or protein fragment) that demonstrates aninteraction with the soluble target may be selected.

In several embodiments, a known or particularly desirable epitope on thesoluble target is “pre-blocked”, for example by a natural antigen thatbinds that epitope. In some cases, for example, if the target is aligand to a receptor, the target can be pre-incubated with a peptidethat encodes the portion of the receptor that interacts with the ligand.Thus, in several embodiments, the target that is to be panned has thekey site of interest blocked when the candidate molecules are screened.When the candidate molecules are screened against the pre-blockedtarget, a portion of the candidate molecules will bind to the target anda portion will fail to bind to the target. Those that bind to the targetare less desirable than those that fail to bind, as those that bind areinteracting with the target at a site other than the epitope of interest(which is blocked). Thus, in several embodiments, the candidatemolecules that fail to bind the target are collected and those that bindthe target are discarded. It shall be appreciated that this“pre-blocking” step is optional, and multiple rounds of screening ofcandidate molecules can be performed and selection of a final candidatebeing based on other characteristics of the molecule and/or the target.However, advantageously, the pre-blocking and negative selection, inseveral embodiments provide a more efficient screening process andresult in a pool of candidate molecules that are more precisely definedin terms of the epitopes with which they will interact.

In some embodiments, the retained candidate molecules that failed tobind to the pre-blocked epitope of interest are further screened againsta target with an unblocked epitope. These will only bind to the chosenepitope since the proteins that bind to other epitopes have beendiscarded. Such additional screening can be used to provide confirmationof the specificity of the candidate molecules. Competitive bindingassays may also optionally be performed in order to characterize thevarious candidate molecules in terms of their interaction efficiencywith the target of interest.

In FIG. 17B, an antigen with known code for its mRNA is used to immunizepatient(s). The immunized patient makes antibodies to the antigen. Asshown in FIG. 17B, an antigen that is known is selected. In someembodiments, the antigen is one to which a subject has already beenexposed. In some embodiments, the known antigen is administered to thesubject, in order to “vaccinate” the subject and induce the generationof antibodies against that antigen. The advantage of using a knownantigen are that an mRNA sequence known to encode that antigen mayalready be known, thereby reducing the need to pan an additionalprotein-mRNA library to generate the portion of the therapeutic thatwill elicit an immune response. However, in some embodiments, a secondpanning step is used.

In FIG. 17C, the two mRNAs, converted to cDNA and PCR amplified, arethen enzymatically fused with a bridge cDNA between the two cDNAs. Asshown in FIG. 17C, the mRNA encoding the protein that interacts with thesoluble target and the mRNA that encodes the known antigen are, in someembodiments, converted to cDNA by a reverse transcription reaction. Insome embodiments, a linker or bridge sequence of cDNA is ligated betweenthe two cDNAs, and then the entire sequence is amplified by polymerasechain reaction. In some embodiments, the bridge cDNA encodes a sequenceof known hydrophilic amino acids. In some embodiments, the bridge (orother portion of the cDNAs) encode a series of amino acids that can belater used to purify the resultant protein (e.g., a histidine tag,allowing for well-established nickel-sepharose chromatography methods).In some embodiments, the individual cDNAs are first amplified and thenligated with the bridge cDNA. Regardless, of the order of amplificationand ligation, the resultant sequence of cDNA (now comprising a cDNAsequence for the protein that interacts with the soluble target and acDNA sequence that encodes the known antigen and a bridge cDNA sequence)is then, in some embodiments, translated into protein.

In still additional embodiments, the individual cDNAs are translatedinto protein and later linked together to form the bifunctionaltherapeutic. In some embodiments, a protein coupling agent, such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexaned-iamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethyl-enediamine), diisocyanates (such astolyene 2,6-diisocyanate), or bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene), is used to link two or more proteincomponents comprising the therapeutic. In some embodiments, such anapproach is particularly advantageous as a variety of pools of solubleagent binding domains and antigenic domains can be prepared, stored, andlater used in various combinations depending on the needs of aparticular subject. In some embodiments, the translation is in vitro,while in some embodiments, in vivo translation is used, followed byisolation of the resultant protein (e.g., by gel electrophoresis, sizeexclusion, selective tag purification, or any other means known in theart to purify proteins).

After generation of a putative bifunctional therapeutic, a series ofpanning steps are performed, in some embodiments, to identify and selectfor therapeutics that are more efficient at interacting with the solubleagent and/or an antibody. As shown in FIG. 18A, a pool of bifunctionaltherapeutic candidate molecules are panned (e.g., reacted with) thesoluble target of interest, which is bound to a solid substrate. Thecandidate molecules are represented by the small geometric,semi-rectangular geometric shapes (the portion which may interact with asoluble target) a linker portion (the curvy line) and a rounded portion(represents the antigen domain, which will interact with an antibodygenerated by the “vaccinated” subject).

As shown in Panel B, the bifunctional proteins that recognize thesoluble target will bind with it; those that do not will be removed bywashing (or other similar removal step). Those that bind with thesoluble target are collected and the soluble target is removed (e.g., byincubation or washing in detergent-rich buffers), in some embodiments,re-reacted with unbound soluble target. In several embodiments, such anapproach assures that the soluble target does not cause steric hindrancechanges in the bifunctional protein that affect the reaction with theantibody. As shown in Panel C, the target bound bifunctional protein isreacted with antibodies that are directed to the known antigen portionof the bifunctional protein. In several embodiments, the antibodies thatbind the antigen portion of the bifunctional proteins are affixed to asolid substrate. Only bifunctional protein candidates with the correctimmunizing antigen will bind with the idiotype site of the antibodieswhile those with other antigens will not, and can be removed (e.g.,washed) from the reaction site). As represented in Panel D, only thebifunctional proteins with both correct ends are collected. Afterremoval of the soluble target component, these bifunctional proteinsbecome the therapeutic. In other embodiments, other methods are used todetermine if the candidate therapeutics interact with the soluble targetand/or the generated antibody, for example Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate, Fabidiotype fragments, peptides, idiotype-expressing cells or extractsthereof.

Therapeutics with Direct Antibody Binding Regions

As shown in FIGS. 20A-20D, in some embodiments, a bifunctionaltherapeutic that comprises a region that interacts with the solubletarget and a region that binds directly to the structural portions of anantibody are generated. As shown in FIG. 20A, the soluble target onsolid substrate is panned with a protein-SATA-mRNA library under bothneutral and acid conditions to stimulate plasma and endosome conditions,similar to that described above.

As discussed above, in several embodiments, a known or particularlydesirable epitope on the soluble target is “pre-blocked”, for example bya natural antigen that binds that epitope. Thus, in several embodiments,the target that is to be panned has the key site of interest blockedwhen the candidate molecules are screened. When the candidate moleculesare screened against the pre-blocked target, a portion of the candidatemolecules will bind to the target and a portion will fail to bind to thetarget. Those that bind to the target are less desirable than those thatfail to bind, as those that bind are interacting with the target at asite other than the epitope of interest (which is blocked). Thus, inseveral embodiments, the candidate molecules that fail to bind thetarget are collected and those that bind the target are discarded.

In some embodiments, the retained candidate molecules that failed tobind to the pre-blocked epitope of interest are further screened againsta target with an unblocked epitope. Such additional screening can beused to provide confirmation of the specificity of the candidatemolecules prior to generation of a final bifunctional protein.

In FIG. 20B, IgG on solid substrate is panned with protein-SATA-mRNAlibrary under both neutral and acid conditions to simulate plasma andendosome conditions. For both panning methods, both neutral and acidicconditions are used to simulate both plasma and endosome-likeenvironments. In FIG. 20C, the two mRNA's are converted to cDNA and PCRamplified. then enzymatically fused with a bridge cDNA between them. Thefused, cDNA is then PCR amplified and transcribed to mRNA and translatedin vitro, and screened for intact bifunctional proteins.

In some embodiments, the antibody panned is an IgG, though IgM, IgA, orIgE isotypes are used in some embodiments. In some embodiments, usingIgG isotypes, isotype 1, 2, 3, or 4 can be used, depending on theembodiment. In some embodiments, the optimum binding protein-IgG isotypecombinations will be made empirically, while in others, the isotype canbe selected ahead of time. In some embodiments, proteins that bind tothe variable regions are identified by panning and used in generation ofthe bifunctional therapeutic. In several embodiments, those proteinsthat bind to the constant region of an antibody are used. In someembodiments, the constant heavy chain is the preferred binding site ofproteins to be used in generating the therapeutic.

FIG. 21 depicts a representative targeted therapeutic that is consistentwith several embodiments disclosed herein. It shall be appreciated thatother interactions with antibodies are used, depending on the specificembodiment.

Several embodiments utilizing IgG are particularly advantageous due tothe presence of the FcRn binding site on the IgG. The FcRn binding siteis located between the C_(H2) and C_(H3) regions of the Fc stem of theIgG, provides a salvage pathway for returning IgG to the circulation. Assuch, use of IgG antibodies provide a longer residence time in thecirculation, and, in some embodiments, an increased therapeutic time.

As discussed above, the selections, in some embodiments, are performedat both neutral and acid pH (pH 5-6.2) to ensure that the bifunctionalprotein will remain bound to the soluble target while in the endosome.

The remainder of the generation of the targeted therapeutic is performedas described above, with the exception, of course, that one portion ofthe therapeutic will bind directly to a portion of an antibody thatpreexists in a subject. As such, this approach advantageously obviatesthe need for vaccination of the subject with a known antigen prior toadministration of the targeted therapeutic.

In FIG. 22A, bifunctional protein candidates are panned against solubletarget affixed on a solid substrate. In FIG. 22B, the bifunctionalproteins that recognize the soluble target will bind with it, and thosethat do not recognize the soluble target will not bind. Those that bindare collected, and then re-reacted with soluble target. This assuresthat the soluble target does not cause steric hindrance changes in thebifunctional protein that might affect the reaction with the CH1 portionof the antibody. In FIG. 22C, the bifunctional protein candidatespre-reacted with soluble target are panned against IgG affixed to solidsubstrate. Bifunctional protein candidates that recognize the CH1portion of the IgG specifically will bind, and those that do notrecognize the CH1 portion will not bind. In FIG. 22D, only thebifunctional proteins with both correct ends are collected. With removalof the soluble target component, they can become therapeutic.

Screening of candidate bifunctional proteins is likewise performed asdescribed above, with the exception that the antibody that is panned ismatched to the antibody isotype that the mRNA used to generated thebifunctional protein encoded (e.g., IgG with IgG). See, for exampleFIGS. 23A-23E.

Administration of Bifunctional Targeted Therapeutics TherapeuticsExploiting a Known Antigen

In several embodiments, the initiation of therapy with bifunctionaltherapeutics that exploit known antigens occurs when a subject who hasbeen exposed to a soluble target is administered the known antigen. Asdiscussed above, an mRNA that encodes the known antigen will be used inthe generation of the bifunctional therapeutics. The pre-administrationof the known antigen to the subject acts to induce generation ofantibodies directed against that antigen. After the screening of thecandidate bifunctional therapeutics and identification of one (or more)that appropriately react with both the soluble target and the antibody,the identified bifunctional therapeutics are administered to thesubject. FIG. 19A shows the therapeutic bifunctional protein beinginfused into the patient. As shown in FIG. 19B, the “target” side of thebifunctional therapeutic binds with the soluble target. The antigenportion of the bifunctional therapeutic is bound by the patient's ownantibodies against the antigen. As seen in FIG. 19C, the result of thetwo reactions is that the soluble target which is bound indirectly viathe bifunctional therapeutic to the antibodies is flagged fordestruction by the immune system, thereby removing the target from theindividual.

Therapeutics with Direct Antibody Binding Regions

In contrast to the use of the bifunctional therapeutic proteins whereinone portion of the protein comprises an antigen against which thepatient has been immunized, the administration of therapeutics with aregion that directly binds to a portion of an antibody dose not need tobe preceded by vaccination of the subject with a known antigen. Thus, inseveral embodiments, a subject who has been exposed to a soluble targetis identified. As discussed above, a bifunctional therapeutic isgenerated that is directed specifically to the soluble target and alsoto a specific portion of an antibody (e.g., an IgG). After screening thecandidate pool, the selected bifunctional therapeutics are administeredto a subject. In the subject's circulation, the target portion of thebifunctional protein interacts with the soluble target. Similarly, theantibody binding portion interacts with a circulating antibody. Inseveral embodiments, the antibody binding portion binds to the heavychain of an IgG within the constant region (e.g., the C_(H1) bindingsite). The schematic for the subsequent reaction in vivo is illustratedin FIG. 23 . As shown in FIG. 23A, macrophages express Fc receptors ontheir surface. In several embodiments, after administration of thebifunctional therapeutic, the Fc domain of the IgG complexes bound tothe bifunctional therapeutic will bind the Fc receptors on themacrophages (FIG. 23B). The bifunctional complex is subsequentlyphagocytosed (FIG. 23C). After phagocytosis, the bifunctionaltherapeutic will be digested to small peptide fragments by enzymes inthe endosome, lysosome or other acid vesicle to which the bifunctionaltherapeutic is trafficked. While in the lysosome (or other vesicle), inseveral embodiments, the resultant fragments are associated with MHC IIand are subsequently presented on the surface of the macrophage. Once onthe surface, they are recognized by CD4⁺ T cells that express theappropriate T cell receptor. Binding of the fragment, which is nowfunctionally an antigen, and CD4 to the MHC II activates the T cells tosecrete cytokines (interleukins) that activate B cells. The activated Bcells undergo proliferation and produce antibodies against the antigenfragment. Thus, in several embodiments, the single administration of thebifunctional therapeutic induces a last therapeutic immune response.However, in some embodiments, both for the direct antibody bindingtherapeutic and the antigen encoding (pre-immunization) therapeutic,two, three, four, five, or more administrations may be given in certainembodiments.

While a number of preferred embodiments of the current invention andvariations thereof have been described in detail, other modificationsand methods of use will be readily apparent to those of skill in theart. For all of the embodiments described above, the steps of themethods need not be performed sequentially. Accordingly, it should beunderstood that various applications, modifications and substitutionsmay be made without departing from the spirit of the invention or thescope of the claims.

Example 1: Production of the SATA

One skilled in the art will understand that the SATA can be produced ina number of different ways. The protocols described below in thefollowing examples can be used for SATAs that have both a puromycin anda crosslinker on the tRNA, or that have a puromycin on the tRNA and acrosslinker on the mRNA. Where the crosslinker is on the mRNA, Example4, below, provides guidance. The following protocol is also instructivefor Linking tRNA Analogs, in the sense that Linking tRNA Analogs also,in a preferred embodiments, have a crosslinker on the tRNA.

For example, in a preferred embodiment, three fragments (FIG. 1 ) werepurchased from a commercial source (e.g., Dharmacon Research Inc.,Boulder, Colo.). Modified bases and a fragment 3 with a pre-attachedpuromycin on its 3′ end and a PO4 on its 3′ end were included, all ofwhich were available commercially. Three fragments were used tofacilitate manipulation of the fragment 2 in forming the monoadduct.

Yeast tRNAAla or yeast tRNAPhe were used; however, sequences can bechosen from widely known tRNAs or by selecting sequences that will forminto a tRNA-like structure. Preferably, sequences with only a limitednumber of U's in the portion that corresponds to the fragment 2 areused. Using a sequence with only a few U's is not necessary becausepsoralen preferentially binds 5′UA3′ sequences (Thompson J. F., et alBiochemistry 21:1363, herein incorporated by reference). However, therewould be less doubly adducted product to purify out if such a sequencewas used.

Fragment 2 was preferably used in a helical conformation to induce thepsoralen to intercalate. Accordingly, a complementary strand wasrequired. RNA or DNA was used, and a sequence, such as poly C to one orboth ends, was added to facilitate separation and removal aftermonoadduct formation was accomplished.

Fragment 2 and the cRNA were combined in buffered 50 mM NaCl solution.The Tm was measured by hyperchromicity changes. The two molecules werereannealed and incubated for 1 hour with the selected psoralen at atemperature˜10° C. less than the Tm. The psoralen was selected basedupon the sequence used. A relatively insoluble psoralen, such as 8 MOP,could be selected which has a higher sequence stringency but may need tobe replenished. A more soluble psoralen, such as AMT, has lessstringency but will fill most sites. Preferably, HMT is used. If afragment 2 is chosen that contains more non-target U's, a greaterstringency is desired. Decreasing the temperature or increasing ionicstrength by adding Mg++ was also used to increase the stringency. In apreferred embodiment, MG++ was omitted and ˜400 mM NaCl solution wasused.

Following incubation, psoralen was irradiated at a wavelength greaterthan approximately 400 nm. The irradiation depends on the wavelengthchosen and the psoralen used. For instance, approximately 419 nm 20-150J/cm2 was preferably used for HMT. This process results in an almostentirely furan sided monoadduct.

Purification of a Monoadduct

The monoadduct was then purified by HPLC as described in Sastry et al,J. Photochem. Photobiol. B Biol. 14:65-79, herein incorporated byreference. The fact that fragment 2 was separate from fragment 3facilitated the purification step because, generally, purification ofmonoadducts≥25 mer is difficult (Spielmann et al. PNAS 89: 4514-4518,herein incorporated by reference).

Ligation of Fragment 2 and 3

The fragment 2 was ligated to the fragment 3 using T4 RNA ligase. Thepuromycin on the 3′ end acted as a protecting group. This is done as perRomaniuk and Uhlenbeck, Methods in Enzymology 100:52-59 (1983), hereinincorporated by reference. Joining of fragment 2+3 to the 3′ end offragment 1 was done according to the methods described in Uhlenbeck,Biochemistry 24:2705-2712 (1985), herein incorporated by reference.Fragment 2+3 was 5′ phosphorylated by polynucleotide kinase and the twohalf molecules were annealed.

In an alternative method, significant quantities of furan sidedmonoadducted U were formed by hybridizing poly UA to itself andirradiating as above. The poly UA was then enzymatically digested toyield furan sided U which was protected and incorporated into a tRNAanalog by nucleoside phosphoramidite methods. Other methods of formingthe psoralen monoadducts include the methods described in Gamper et al.,J. Mol. Biol. 197: 349 (1987); Gamper et al., Photochem. Photobiol.40:29, 1984; Sastry et al, J. Photochem. Photobiol. B Biol. 14:65-79;Spielmann et al. PNAS 89:4514-4518, U.S. Pat. No. 4,599,303, all hereinincorporated by reference.

SATAs generated by the methods described above read UAG (anticodon CUA).Additionally, UAA or UGA was also used. In various embodiments, anymessage that had the stop codon that was selected as the “linking codon”was used.

Example 2: Production of Psoralenated Furan Sided Monoadducts UV LightExposure of RNA:DNA Hybrids

Equal volumes of 3 ng/ml RNA:cRNA hybrid segments and of 10 μg/ml HMTboth comprised of 50 mM NaCl were transferred into a new 1.5 ml cappedpolypropylene microcentrifuge tube and incubated at 37° C. for 30minutes in the dark. This was then transferred onto a new clean culturedish. This was positioned in a photochemical reactor (419 nm peakSouthern New England Ultraviolet Co.) at a distance of about 12.5 cm sothat irradiance was ˜6.5 mW/cm2 and irradiated for 60-120 minutes.

Removal of Low Molecular Weight Protoproducts

100 μl of chloroform-isoamyl alcohol (24:1) was pipetted and mixed byvortex. The mixture was centrifuged for 5 minutes at 15000×g in amicrocentrifuge tube. The chloroform-isoamyl alcohol layer was removedwith a micropipette. The chloroform-isoamyl alcohol extraction wasrepeated once again. Clean RNA was precipitated out of the solution.

Alcohol Precipitation

Two volumes (˜1000 μl) ice cold absolute ethanol was added to themixture. The tube was centrifuged for 15 minutes at 15,000×g in amicrocentrifuge. The supernatant was decanted and discarded and theprecipitated RNA was redissolved in 100 μl DEPC treated water thenre-exposed to the RNA+8-MOP.

Isolation of the Psoralenated RNA Fragments Using HPLC

All components, glassware and reagents were prepared so that they wereRNAase free. The HPLC was set up with a Dionex DNA PA-100 packagecolumn. The psoralenated RNA:DNA hybrid was warmed to 4° C. Thepsoralenated RNA was applied to HPLC followed by oligonucleotideanalysis, as described in the following section entitled“Oligonucleotide Analysis by HPLC.” The collected fractions represented:

(SEQ ID NO: 1) 5′CUAGAΨCUGGAGG3′, where Ψ is pseudouridine (SEQ ID NO:2) Furan sided 5′CUPsoralenAGAΨCUGGAGG3′ monoadducts (SEQ ID NO: 3)5′XXXXXCCUCCAGAUCUAGXXXXX3′ (SEQ ID NO: 4)5′XXXXXCCUCCAGAUCUPsoralenAGXXXXX3′

The fractions were stored at 4° C. in new, RNAase free snappedmicrocentrifuge tubes and stored at −20° C. if more than four weeks ofstorage were required.

Identification of the RNA Fragments Represented by Each Peak FractionCollected by HPLC Using Polyacrylamide Gel Electrophoresis (PAGE)

The electrophoresis unit was set up in a 4° C. refrigerator. A gel wasselected with a 2 mm spacer. Each 5 μl of HPLC fraction was diluted to10 μl with Loading Buffer. 10 μl of each diluted fraction was loadedinto appropriately labeled sample wells. The tracking dye was loaded ina separate lane and electrophoresis was run as described in thefollowing section entitled “Polyacrylamide Gel Electrophoresis (PAGE) ofPsoralenated RNA Fragments.” After the electrophoresis run was complete,the electrophoresis was stopped when the tracking dye reached the edgeof the gel. The apparatus was disassembled. The gel-glass panel unit wasplaced on the UV light box. UV lights were turned on. The RNA bands wereidentified. The bands appeared as denser shadows under UV lightingconditions.

Extraction of the RNA from the Gel

Each band was excised with a new sterile and RNAase free scalpel bladeand transferred into a new 1.5 ml snap capped microcentrifuge tube. Eachgel was crushed against the walls of the microcentrifuge tubes with theside of the scalpel blade. A new blade was used for each sample. 1.0 mlof 0.3 M sodium acetate was added to each tube and eluted for at least24 hours at 4° C. The eluate was transferred to a new 0.5 ml snap cappedpolypropylene microcentrifuge tube with a micropipette. A new RNAasefree pipette tip was used for each tube and the RNA with ethanol wasprecipitated out.

Ethanol Precipitation

Two volumes of ice cold ethanol was added to each eluate thencentrifuged at 15,000×g for 15 minutes in a microcentrifuge. Thesupernatants were discharged and the precipitated RNA was re-dissolvedin 100 μl of DEPC treated DI water. The RNA was stored in themicrocentrifuge tubes at 4° C. until needed. The tubes were stored at−20° C. if storage was for more than two weeks. The following was orderof rate of migration for each fragment in order from fastest to slowest:

(SEQ ID NO: 1) 5′CUAGAΨCUGGAGG3′ (SEQ ID NO: 2) Furan sided5′CUPsoralenAGAΨCUGGAGG3′ monoadducts (SEQ ID NO: 3)5′ XXXXXCCUCCAGAUCUAGXXXXX3′ (SEQ ID NO: 4)5′ XXXXXCCUCCAGAUCUPsoralenAGXXXXX3′

The tubes containing the remainder of each fraction were labeled andstored at −20° C.

Ethanol Precipitation

RNA oligonucleotide fragments were precipitated, and all glassware wascleaned to remove any traces of RNase as described in the followingsection entitled “Inactivation of RNases on Equipment, Supplies, and inSolutions.” All solutions were stored in RNAase free glassware andintroduction of nucleases was prevented. Absolute ethanol was stored at0° C. until used. Micropipettes were used to add two volumes of ice coldethanol to nucleic acids that were to be precipitated in microcentrifugetubes. Capped microcentrifuge tubes were placed into the microfuge andspun at 15,000×g for 15 minutes. The supernatant was discarded andprecipitated RNA was re-dissolved in DEPC treated DI-water. RNA wasstored at 4° C. in microcentrifuge tubes until ready to use.

Ligation of RNA Fragments 2 and 3

All glassware was cleaned to remove any traces of RNase as described inthe following section entitled “Inactivation of RNases on Equipment,Supplies, and in Solutions.” The following was added to a new 1.5 mlpolypropylene snap capped microcentrifuge tube using a 100-1000 μlpipette and a new sterile pipette tip was used for each solution:

Fragment 2 (3.0 nM) 125.0 μl Fragment 3 (3.0 nM) 125.0 μl Reactionbuffer 250.0 μl RNA T4 ligase (9-12 U/ml)   42 μl

Reaction Buffer

RNase free DI-water 90.00 ml Tris-HCl (50 mM) 0.79 g MgCl2 (10 mM) 0.20g DTT (5 mM) 0.078 g ATP (1 mM) 0.55 g pH to 7.8 with HCL RNase freeDI-water QS to 100.00 ml

The mixture was gently mixed and the RNA was melted by incubating themixture at 16° C. for one hour in a temperature controlled refrigeratedchamber. RNA was precipitated out of the solution immediately after theincubation was completed.

Alcohol Precipitation

Two volumes (˜1000 μl) of ice cold absolute ethanol were added to thereaction mixture. The microcentrifuge tube was placed in amicrocentrifuge at 15,000×g for 15 minutes. The supernatant was decantedand discarded and the precipitated RNA was redissolved in 100 μl DEPCtreated water. The mixture was electrophoresed as described in thefollowing section entitled “Polyacrylamide Gel Electrophoresis (PAGE) ofPsoralenated RNA Fragments.” The following was the order of rate ofmigration for each fragment in order from fastest to slowest:

a) Frag. 2 (SEQ ID NO: 5) 5′CUAGAΨCUGGAGG3′-OHPsoralen b) Frag. 3 (SEQID NO: 6) 5′UCCUGUGTΨCGAUCCACAGAAUUCGCACC-Puromycin c) Frag 2 + 3 (SEQID NO: 7) 5′CUPsoralenAGAYCUGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCA CCPuromycin

Each fraction was isolated by UV shadowing, the bands were cut out, theRNAs were eluted from the gels and the RNA elute was precipitated out asdescribed in the following section entitled “Polyacrylamide GelElectrophoresis (PAGE) of Psoralenated RNA Fragments.” The ligationprocedure was repeated with any residual unligated fragment 2 and 3fractions. The ligated fractions 2 and 3 were pooled and stored in asmall volume of RNase free DI-water at 4° C.

Ligation of RNA Fragment 1 with Fragment 2+3

All glassware was cleaned to remove any traces of RNase as described inthe following section entitled “Inactivation of RNases on Equipment,Supplies, and in Solutions.” The following was added to a new 1.5 mlpolypropylene snap capped microcentrifuge tube. A 100-1000 μl pipetteand new tip was used for each solution:

Fragment 2 + 3 (3.0 nM) 125.0 μl Reaction buffer 250.0 μl T4Polynucleotide Kinase(5-10 U/ml)  1.7 μl

Reaction Buffer

RNase free DI-water 90.00 ml Tris-HCl (40 mM) 0.63 g MgCl2 (10 mM) 0.20g DTT (5 mM) 0.08 g ATP (1 mM) 0.006 g pH to 7.8 with HCL RNase freeDI-water QS to 100.00 ml

The RNA was gently mixed then melted by heating the mixture to 70° C.for 5 minutes in a heating block. The mixture was cooled to roomtemperature over a two hour period and the RNA was allowed to anneal ina tRNA configuration. The RNA was precipitated out of the solution.

Alcohol Precipitation

Two volumes (˜1000 μl) of ice cold absolute ethanol were added to thereaction mixture. The microcentrifuge tube was placed in amicrocentrifuge at 15,000×g for 15 minutes. The supernatant was decantedand discarded and the precipitated RNA was redissolved in 100 μl DEPCtreated water. The mixture was electrophoresed as described in thefollowing section entitled “Polyacrylamide Gel Electrophoresis (PAGE) ofPsoralenated RNA Fragments.” The following was the order of rate ofmigration for each fragment in order from fastest to slowest:

a) Frag. 1 (SEQ ID NO: 8) 5′GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACU3′ b) Frag2 + 3 (SEQ ID NO: 6) 5′CUPsoralenAGAYCUGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCACCPuromycin c) Frag. 1 + 2 + 3 (SEQ ID NO: 9)5′GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACUCUPsoralenAGAΨCUGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCACCPuromycin

Each fraction was isolated by UV shadowing, the bands were cut out, theRNAs were eluted from the gels and the RNA elute was precipitated out asdescribed in the following section entitled “Polyacrylamide GelElectrophoresis (PAGE) of Psoralenated RNA Fragments.” The ligationprocedure was repeated with the unligated Fragment 1 and the 2+3Fraction. The ligated fractions 2+3 were pooled and stored in a smallvolume of RNase free DI-water at 4° C.

Final RNA Ligation

The following was added to a new 1.5 ml polypropylene snap cappedmicrocentrifuge tube. A 100-1000 μl pipette and new tip was used foreach solution:

Fragment 1 + 2 + 3 (3.0 nM) 250 μl reaction buffer 250 μl RNA T4 ligase(44 μg/ml) 22 μg

The mixture was incubated at 17° C. in a temperature controlledrefrigerator for 4.7 hours. Immediately after the incubation the tRNAwas precipitated out as described in step 6.2 above and the tRNA wasisolated by electrophoresis as described in the following sectionentitled “Polyacrylamide Gel Electrophoresis (PAGE) of Psoralenated RNAFragments.” The tRNA was pooled in a small volume of RNase free waterand stored at 4° C. for up to two weeks or stored at −20° C. for periodslonger than two weeks.

Polyacrylamide Gel Electrophoresis (Page) of Psoralenated RNA FragmentsAcrylamide Gel Preparation

All reagents and glassware were made RNAase free as described in thefollowing section entitled “Inactivation of RNases on Equipment,Supplies, and in Solutions.” The gel apparatus was assembled to producea 4 mm thick by 20 cm×42 cm square gel. 29 parts acrylamide with 1 partammonium crosslinker were mixed at room temperature with the appropriateamount of acrylamide solution in an RNAase free, thick walled Erlenmeyerflask.

Acrylamide Solution

urea (7M) 420.42 g TBE (1X) QS to 1 L 5X TBE 0.455M Tris-HCl 53.9 g 10mM EDTA 20 ml of 0.5M RNAase free DI water 900 ml pH with boric acid topH 9 QS with RNAase free DI water to 1 L

The mixture was degassed with vacuum pressure for one minute. Theappropriate amount of TEMED was added, mixed gently, and then the gelmixture was poured between the glass plates to within 0.5 cm of the top.The comb was immediately inserted between the glass sheets and into thegel mixture. An RNAase free gel comb was used. The comb produced wellsfor a 5 mm wide dye lane and 135 mm sample lanes. The gel was allowed topolymerize for about 30-40 minutes then the comb was carefully removed.The sample wells were rinsed out with a running buffer using amicropipette with a new pipette tip. The wells were then filled withrunning buffer.

Sample Preparation

An aliquot of the sample was suspended in loading buffer in a snapcapped microcentrifuge tube and vortex mixed. Indicator dye was notadded to the sample.

Loading Buffer

Urea (7M) 420.42 g Tris HCl (50 mM) 7.85 g QS with RNAase free D-H2O to1 L

Electrophoresis Run

The maximum volume of RNA/loading buffer solution was loaded into the135 mm sample wells and the appropriate volume of tracking dye in 5 mmtracking lane. The samples were electrophoresed in a 5° C. refrigerator.The electrophoresis was stopped when the tracking dye reached the edgeof the gel. The apparatus was then disassembled. Glass panels were notremoved from the gel. The gel-glass panel unit was placed on a UV lightbox. With UV filtering goggles in place, the UV lights were turned on.The RNA bands were identified. They appeared as denser shadows under UVlighting conditions. The RNA was extracted from the gel. Each band wasexcised with a new sterile and RNAase free scalpel blade and each bandwas transferred into a new 1.5 ml snap capped microcentrifuge tube. Eachgel was crushed against the walls of the microcentrifuge tubes with theside of the scalpel blade. A new blade was used for each sample. 1.0 mlof 0.3M sodium acetate was added to each tube and eluted for at least 24hours at 4° C. The eluate was transferred to a new 0.5 ml snap cappedpolypropylene microcentrifuge tubes with a micropipette with a newRNAase free pipette tip for each tube. Two volumes of ice cold ethanolwas added to each eluate, then centrifuged at 15,000×g for 15 minutes ina microcentrifuge. The supernatants were discarded and the precipitatedRNA was redissolved in 100 μl of DEPC treated DI water. The RNA wasstored in the microcentrifuge tubes at 4° C. until needed.

Oligonucleotide Analysis By HPLC

HPLC purification of the RNA oligonucleotides is best effected usinganion exchange chromatography. Either the 2′-protected or 2′-deprotectedforms can be chromatographed. The 2′-protected form offers the advantageof minimizing secondary structure effects and provides resistance tonucleases. If the RNA is fully deprotected, sterile conditions arerequired during purification.

Deprotection of 2′-Orthoester Protected RNA

The tubes are centrifuged at 15,000×g for 30 seconds or until the RNApellet is at the bottom. 400 μl of pH 3.8 deprotection buffer is addedto each tube of RNA.

Deprotection Buffer

Acetic acid (100 mM) is adjusted to pH 3.8 withtetramethylethylenediamine (TEMED). The pellet is completely dissolvedin the buffer by drawing in and out of a pipette. The tubes are vortexedfor 10 seconds and centrifuged at 15,000×g. The tubes are incubated in a60° C. water bath for 30 minutes. The samples are lyophilized beforeuse.

HPLC Column Conditions

A 4×250 mm column (DNAPAC PA, No. 043010) packed with Dionex(800)-DIONEX-0 (346-6390), with a capacity of 40 optical density units(ODU) at 260 nm is installed. The column temperature is set to 54° C.The injection volume is adjusted such that 5 μl produces approximately0.20 ODU.

Elution Buffers

Condition Buffer A Buffer B Sodium perchlorate (5 mM) 2.8 g (300 mM)168.0 g Tris-HCl 2.4 g 2.4 g Acetonitrile (2%) 80.0 ml 80.0 ml DI Water3900 ml 900 ml Adjusted pH 8.0 with HCL 8.0 with HCL q.s. 4000 ml 4000ml

HPLC Gradient

A 30% to 60% gradient of Buffer B for oligos 17-32 base pairs long isprovided:

Time Flow (minutes) (ml/min) % A % B Curve 0 1.5 100 0 * 1 1.5 100 0 6 31.5  70* 30* 6 15 1.5  40* 60* 6 15.5 2.5  0 100  6 17 2.5  0 100  617.25 2.5 100 0 6 23 2.5 100 0 6 23.1 1.5 100 0 6 24 1.5 100 0 6 25 0.1100 0 6 *% values that can be changed to modify the gradient. Typicalgradients are 0-30%, 20-50%, 30-60%, and 40-70% of Buffer B.

Gradient Selection

The gradient is selected based upon the number of bases, as follows:

Number of bases Gradient 0-5  0-30  6-10 10-40 11-16 20-50 17-32 30-6033-50 40-70 >50 50-80

After HPLC, the target samples are collected and the RNA concentrationis determined with a spectrophotometer at 260 nm. The samples are storedat −70° C.

Inactivation of RNAses on Equipment, Supplies, and in Solutions

Glassware was treated by baking at 180° C. for at least 8 hours.Plasticware was treated by rinsing with chloroform. Alternatively, allitems were soaked in 0.1% DEPC.

Treatment with 0.1% DEPC

0.1% DEPC was prepared. DI water was filtered through a 0.2 μM membranefilter. The water was autoclaved at 15 psi for 15 minutes on a liquidcycle. 1.0 g (wt/v) DEPC/liter of sterile filtered water was added.

Glass and Plasticware

All glass and plasticware was submerged in 0.1% DEPC for two hours at37° C. The glassware was rinsed at least 5× with sterile DI water. Theglassware was heated to 100° C. for 15 minutes or autoclaved for 15minutes at 15 psi on a liquid cycle.

Electrophoresis Tanks Used for Electrophoresis of RNA

Tanks were washed with detergent, rinsed with water then ethanol and airdried. The tank was filled with 3% (v/v) hydrogen peroxide (30 ml/L) andleft standing for 10 minutes at room temperature. The tank was rinsed atleast 5 times with DEPC treated water.

Solutions

All solutions were made using Rnase free glassware, plastic ware,autoclaved water, chemicals reserved for work with RNA and RNase freespatulas. Disposable gloves were used. When possible, the solutions weretreated with 0.1% DEPC for at least 12 hours at 37° C. and then heatedto 100° C. for 15 minutes or autoclaved for 15 minutes at 15 psi on aliquid cycle.

RNA Translation

2 μl of gastroinhibitory peptide (GIP) mRNA at a concentration of 20μl/ml was placed in a 250 μl snapcap polypropylene microcentrifuge tube.35 μl of rabbit reticulocyte lysate (available commercially fromPromega) was added. 1 μl of amino acid mixture which did not containmethionine (available commercially from Promega) was added. 1 μl of ³⁵Smethionine or unlabeled methionine was added. 2 μl of ³²P GIP mRNA orunlabeled GIP mRNA was added. Optionally, 2 ml of luciferase may beadded to some tubes to serve as a control. In a preferred embodiment,luciferase was used instead of GIP mRNA. One skilled in the art willunderstand that indeed any mRNA fragment containing the appropriatesequences may be used.

SATA was added to the experimental tubes. Control tubes which did notcontain SATA were also prepared. The quantity of SATA used wasapproximately between 0.1 μg to 500 μg, preferably between 0.5 μg to 50μg. 1 μl of Rnasin at 40 units/ml was added. Nuclease free water wasadded to make a total volume of 50 μl.

For proteins greater than approximately 150 amino acids, the amount oftRNA may need to be supplemented. For example, approximately 10-200 μgof tRNA may be added. In general, the quantity of the SATA should behigh enough to effectively suppress stop or pseudo stop codons. Thequantity of the native tRNA must be high enough to out compete the SATAwhich does not undergo dynamic proofreading under the action ofelongation factors.

Each tube was immediately capped, parafilmed and incubated for thetranslation reactions at 30° C. for 90 minutes. The contents of eachreaction tube was transferred into a 50 μl quartz capillary tube bycapillary action. The SATA was crosslinked with mRNA by illuminating thecontents of each tube with 2-10 J/cm2˜350 nm wavelength light, as perGasparro et al. (Photochem. Photobiol. 57:1007 (1993), hereinincorporated by reference). Following photocrosslinking, the contents ofeach tube were transferred into a new snapcap microfuge tube. Theribosomes were dissociated by chelating the calcium cations by adding 2μl of 10 mM EDTA to each tube. Between each step, each tube was gentlymixed by stirring each component with a pipette tip upon addition.

The optimal RNA for a translation was determined prior to performingdefinitive experiments. Serial dilutions may be required to find theoptimal concentration of mRNA between 5-20 μg/ml.

SDS-Page electrophoresis was performed on each sample, as describedabove. Autoradiography on the gel was performed, as described bySambrook et. al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed.,Coldspring Harbor Press (1989), herein incorporated by reference.

The above example teaches the production and use of SATA (e.g.,puromycin on tRNA plus crosslinker on the tRNA) and the production anduse of Linking tRNA Analog (e.g., no puromycin, but has crosslinker ontRNA).

In another example, the SATA was produced in a manner similar to theabove methodology, except that uridines were substituted withpseudouridines. Substitution by pseudouridines can also be used withLinking tRNA Analog, as it facilities the formation of crosslinkermonoadduct formation (such as formation of the psoralen monoadduct).This technique is discussed below in Example 2.

Example 3: Production of the SATA Using Pseudouridine

As discussed above, one skilled in the art will appreciate that theSATA, Linking tRNA Analog and Nonsense Suppressor tRNA can be producedin a number of different ways. FIG. 5 shows the chemical structures foruridine and pseudouridine. Pseudouridine is a naturally occurring basefound in tRNA that forms hydrogen bonds just as uridine does, but lacksthe 5-6 double bond that is the target for psoralen. Pseudouridine, asused herein, shall include the naturally occurring base and anysynthetic analogs or modifications. In a preferred embodiment, the SATAwas produced using pseudouridine. Linking tRNA Analog can also beproduced using pseudouridine. Specifically, in a preferred embodiment,three fragments (FIG. 1 ) were purchased from a commercial source(Dharmacon Research Inc., Boulder, Colo.). Modified bases and a fragment3 (“Fragment 3”) with a pre-attached puromycin on its 3′ end and a PO₄on its 3′ end were included, all of which are available commercially.The three fragments were used to facilitate manipulation of a fragment 2(“Fragment 2”) in forming the monoadduct. Sequences of the threefragments, according to some embodiments, are as follows (2 examplesequences are provided for each fragment):

Fragment 1 (SEQ ID NO: 10) 5′PO₄GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACOH3′(SEQ ID NO: 16) 5′PO₄GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACOH3′ Fragment 2(SEQ ID NO: 11) 5′OHΨCUAACΨCOH3′ (SEQ ID NO: 17) 5′ OHΨCUAAAΨCOH3′Fragment 3 (SEQ ID NO: 12)5′PO₄UGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCACCPuromycin3′ (SEQ ID NO: 18)5′PO₄UGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCACCPuromycin3′

The above sequences listed in Fragment 3 are applicable for SATA. ForLinking tRNA Analogs, the sequences would be similar, except thepuromycin would be replaced by adenosine.

Modified yeast tRNAAla or yeast tRNAPhe was used according to oneembodiment of the invention. However, one skilled in the art willunderstand that sequences can be chosen widely from known tRNAs or byselecting sequences that will form into a tRNA-like structure. Oneadvantage of using pseudouridine in some embodiments is that thepseudouridine in Fragment 2 avoids psoralen labeling of the nontargetU's. Use of pseudouridine instead of uridine decreases the avidity ofthe A site of the ribosome for the tRNA analog but eliminates theinteraction of the terminal uridine with psoralen. The use of the Yarus“extended anticodon” guidelines increases A site binding (Yarus, Science218:646-652, 1982, herein incorporated by reference).

In one embodiment, Fragment 2 was used in a helical conformation toinduce the psoralen to intercalate. One skilled in the art willunderstand that other conformations can also be used in accordance withseveral embodiments of the invention. A complementary strand was alsoused. RNA or DNA was used, and a sequence, such as poly C or poly G whenC interacts with the psoralen to one or both ends, was added tofacilitate separation and removal after monoadduct formation wasaccomplished. Use of pseudouridine instead of uridines in the complementpermitted the use of a high efficiency wave length, such as about 365nm, without fear of crosslinking the product. Irradiation was preferablyin the range of about 300-450 nm, more preferably in the range of about320 to 400 nm, and most preferably about 365 nm. Further, use ofpseudouridine left the furan-sided monoadduct in place on Fragment 2because the Maf is the predominate first step in the crosslinkformation.

The following cRNA sequences with pseudouridine were used according to apreferred embodiment of the present invention. One skilled in the artwill understand that substitutions and modifications of these sequences,and of the other sequences listed herein, can also be used in accordancewith several embodiments of the current invention. For example, for SEQID NO: 19, listed below, the sequence can also be

(SEQ ID NO: 30) 5′XXXXXXGAΨΨΨAGAXXXXXXX3′: (SEQ ID NO: 13)CCCΨCCAGAGΨΨAGACCC (SEQ ID NO: 19) 5′CCCCCCGAΨΨΨAGACCCCCCC3′

Step 1: Furan Sided Monoadduction of Psoralen to Fragment 2

The formation of a furan sided psoralen monoadduct with the targeturidine of Fragment 2 was performed as follows:

A reaction buffer was prepared as follows:

Tris HCL  25 mM NaCl 100 mM EDTA 0.32 mM  pH 7.0

4′hydroxy methyl-4,5′,8′-triethyl psoralen (HMT) was then added to afinal concentration of 0.32 mM and equimolar amounts of fragment 2 andcRNA were added to a final molar ratio of fragment2:cRNA:psoralen=1:1:1000. A total volume of 100 μl was irradiated at atime.

The mixture of complementary oligos, HMT, psoralen was processed asfollows:

1) Heated to 85° C. for 60 sec followed by cooling to 4° C. over 15 min,using PCR thermocycler.

2) Irradiated for 20 min at 4° C., in Eppendorf UVette plastic cuvette,covered top with parafilm, laid on the top of UV lamp (1 mW/cm²multi-wavelength UV lamp (λ>300 nm) (UV L21 model λ 365 nm).

Steps 1 and 2 above were repeated 4 times to re-intercalate andirradiate HMT. After the second irradiation additional 10 μl of 1.6 mMHMT was added in total 100 μl reaction volume. After 4 cycles ofirradiation, the free psoralens were extracted with chloroform and alloligos (labeled and unlabeled) were precipitated with ethanol overnight(see precipitation step). A small aliquot was saved for gelidentification.

Step 2: Purification of HMT Conjugated Fragment 2 (2 MA) Oligo by HPLC

1) The reaction mixture was dried with speed vacuum for 10 minutes andthen was dissolved with 2 μl of 0.1 M TEAA, pH 7.0 buffer.

0.1 M TEAA, pH 7.0 Buffer

Acetic Acid  5.6 ml Triethylamine 13.86 ml   H₂0 (RNAase free) 950 ml

pH adjusted to 7.0 with acetic acid

and water added to 1 L

2) The sample was loaded onto a Waters Xterra MS C18, 2.5 μm, 4.5×50 mmreverse-phase column pre-equilibrated with buffer A (5% wt/wtacetonitrile in 0.1M TEAA, pH 7.0) The sample was eluted with a gradientof 0-55% buffer B (15% wt/wt acetonitrile in 0.1M TEAA, pH 7.0) tobuffer A over a 35 minute time frame at a flow rate of 1 ml/minute. Thecolumn temperature was 60° C. and the detection wave length, set by anarrow band filter, was 340 nm. Furan sided psoralen monoadduct absorbsat 340 nm but the RNA, and any pyrone sided monoadduct does not. Thebuffer solutions were filtered and degassed before use.

The 2 MA eluted at around 25-28 minutes at a buffer B concentration of40%. Unpsoralenated fragment 2 eluted before 8 minutes based onsubsequent gel electrophoresis analysis on collected fractions.

The column was washed with 100% acetonitrile for 5 minutes and wasre-equilibrated with buffer A for 15 minutes. All fractions were driedwith speed vacuum overnight.

The fractions containing the 2 MA were identified by the level ofabsorbance at 260 nm (RNA) and 330 nm (furan sided psoralen monoadductedRNA). This was done by redissolving the dried fractions with 120 μl ofRnase-free distilled water and the absorbance was measured with aspectrophotometer at 260 nm and 330 nm. The fractions with highabsorbance at both wavelengths were pooled then dried with speed vacuum.A small aliquot from each was saved for gel analysis.

The cross-linked products were analyzed on a denaturing 20% TBE-urea geland visualized by gel silver staining.

Step 3: Purification of HMT Conjugated Fragment 2 Oligo from cRNA byHPLC

The dried samples were pooled and then were dissolved with 0.5×TEbuffer. A sample of about 0.4 absorbance unit was loaded onto a DionexDNAPac PA-100 (4×250 mm) column which was pre-equilibrated with buffer C(25 mM Tris-HCl, pH 8.0) and the column temperature was 85° C. (anionexchange HPLC).

The oligos were eluted at a flow rate of 1 ml/min. with a concavegradient from 4% to 55% buffer D for 15 minutes followed by a convexgradient from 55% to 80% with buffer D for the next 15 minutes. Theoligos were washed with 100% buffer D for 5 min and 100% buffer C foranother 5 min at a flow rate of 1.5 ml/min; Fractions were collectedthat absorbed 260 nm light. 2 MA had a retention time (RT) of 16.2minutes and was eluted by 57% buffer D, and free fragment 2 had RT lessthan 16.6 minutes, and was eluted by 55% buffer D and free cRNA had RTgreater than 19.2 minutes. The fractions were collected that absorbed at254 or 260 nm. The collected fractions were dried with speed vacuumovernight. All solutions were filtered and degassed before use.

The solution used comprised the following:

C: 25 mM Tris-HCl pH 8.0;

D: 250 mM NaClO4 in 25 mM Tris pH 8.0 buffer.

TE: 10 mM Tris-HCl pH 8.0 with 1 mM EDTA

Step 4: Desalting, Precipitation and Collection of the Purified 2 MAOligo

The dried fractions were redesolved with 100 μl Rnase free distilledwater. 500 μl cool 100% ethanol with 0.5M (NH4)2CO3 was added and themixture was vortexed briefly. The mixture was then frozen on dry ice for60 minutes or stored at −20° C. overnight.

The samples were then brought to 4° C. and centrifuged at maximum speedin a microcentrifuge for 15 minutes. The position of the pellet wasnoted and the supernatant was decanted or removed by pipette. Care wastaken not to disturb pellet. If the pellet still contained salt, thisstep was repeated. The pellet was then washed with 70% pre-cooledethanol twice. The wet pellet was dried with speed vacuum for 15 min.Urea PAGE gel identified the right fractions for the next step.

Step 5: Ligation of 2 MA Oligo to Fragment 3 Oligo

The following steps were performed:

A. The following reagents and instruments were used:

Nuclease-Free Water (Promega)

polyethylene glycol (PEG8000 Sigma) 40% (wt/wt in water)

RNasin® Ribonuclease Inhibitor (Promega)

phenol:chloroform

1.5 ml sterile microcentrifuge tubes

100% ethanol

70% ethanol

Dry ice or −20° C. freezer

Microcentrifuge at room temperature and +4° C.

PCR thermocycler or water bath

B. The following reaction conditions were used:

50 mM Tris-HCl (pH 7.8)

10 mM MgCl2,

10 mM DTT

1 mM ATP

18-20% PEG

C. The following reaction mixture was assembled in a sterilemicrocentrifuge tube:

Fragment 3 (Donor) 1 μl (6 μg) (Purified, when necessary, before usingas a donor)

2 MA (Acceptor) 1 μl (1.5 μg)

After adding 8 μl Rnase free dH2O 8 μl, the reactions were incubated at85° C. for 1 minute to relax the oligo secondary structure, then slowlycooled to 4° C., using a PCR machine thermocycler. The preheated tubewas placed on ice to keep cool and centrifuged briefly, then thefollowing was added:

10X Ligase Buffer 4 μl 10 mM ATP 4 μl Rnase Out or Rnasin (40 u/μl)Promega 0.5 μl   PEG, 40% (Sigma) 20 μl  T4 RNA Ligase (10 u/μl) (NEB) 1μl

Nuclease-free water was added to final Volume of 40 μl. The mixture wasincubate at 16° C. overnight (16 hr). The mixture was centrifugedbriefly and then was placed on ice.

D. Precipitation of Oligonucleotides:

60 μl DEPC RNase free distilled water was added to the mixture and then150 μl phenol/chloroform was added. The mixture was vortexed vigorouslyfor 30 seconds. The precipitate was then centrifuged out at maximumspeed in a microcentifuge for 5 minutes at room temperature. The aqueousphase was transferred to a new microcentrifuge tube (>95 μl).

To this was added 3 μl 5 mg/ml glycogen, and 500 μl pre-cooled 100%ethanol with 0.5M (NH4)2CO3 and the mixture was vortexed briefly andthen was frozen on dry ice for 60 minutes. At this point, it may bestored overnight at −20° C. The dried fractions were redissolved with100 μl Rnase-free distilled water, 500 μl cool 100% ethanol with 0.5M(NH4)2CO3 was added and vortexed briefly. This was then frozen on dryice for 60 minutes or stored at −20 C overnight. The samples were thenbrought to 4° C. and centrifuged at maximum speed in a microcentrifugefor 15 minutes and supernatant removed by pipette. Care was taken not todisturb pellet. If the pellet still contained salt, this step wasrepeated once. The pellet was then washed with 70% pre-cooled ethanolseveral times. This was then centrifuged at maximum speed in amicrocentrifuge for 5 minutes at 4 C. The ethanol was carefully removedusing a pipette. Centrifugation was repeated again to collect remainingethanol which was carefully removed. The wet pellet was dried with speedvacuum for 10 min. A small aliquot was collected for the gel analysis.For long term storage, the RNA was stored in ethanol at −20 C. Care wastaken not to store the RNA in DEPC water.

Step 6: Purification of the Ligated Fragment 3 Oligo Complex

The dried sample was redissolved with 0.5×TE buffer and was loaded ontoa DNAPac PA-100 column which was equilibrated with buffer C. The columntemperature was 85° C. and the detector operated at 254 nm to identifyfractions with RNA and at 340 nm to identify fractions with 2 MaF. Theoligos were eluted with a convex gradient from 30% to 70% with buffer Dfor the first 20 minutes at a flow rate of 0.8 ml/min and followed witha linear gradient from 70% to 98% D for another 20 min at the same flowrate. The elution was completed by washing with 100% D for 7 min and100% C for another 10 min at 1.0 ml/min flow rate. The fractions weredetected with 254 or 260 nm wavelength light. The ligated oligos (2MA-fragment 3) were eluted after 34 min, by more than 90% buffer B.Fractions with 254 nm absorbance (A_(254 nm)>0.01) were collected anddried with speed vacuum overnight.

Step 7: Purified 2 MA-Fragment 3 Desalting and Precipitation

The dried fractions were re-dissolved with 100 μl Rnase free distilledwater, 500 μl cool 100% ethanol with 0.5M (NH4)2CO3 was added and themixture was vortexed briefly. The mixture was then frozen on dry ice for60 minutes or stored at −20 C overnight.

The samples were brought to 4° C. and centrifuged at maximum speed in amicrocentrifuge for 15 minutes. The position of the pellet was noted andthe supernatant decanted or removed by pipette. Care was taken not todisturb pellet. If still containing salt, this step was repeated. Thepellet was then washed with 70% pre-cooled ethanol twice. The wet pelletwas dried with speed vacuum for 15 min.

Urea PAGE was performed to identify the ligated 2 MA-fragment-3 for usein the next step of ligating fragment 1 to the 2 MA-fragment-3 oligowhich completes the SATA linker.

Step 8: Preparation of SATA (or Other tRNA Molecule)

A. RNA Oligo 5′phosphorylation

1. Reagent and instrument:

Nuclease-Free Water (Cat. #P1193 Promega)

RNasin® Ribonuclease Inhibitor (Cat #N2511 Promega)

Phenol: chloroform

Sterile microcentrifuge tubes

100% ethanol

70% ethanol

Microcentrifuge at room temperature and 4° C.

PCR thermalcycler or water bath

2. Assemble the following reaction mixture in a sterile microcentrifugetube:

Component Volume Acceptor RNA <200 ng T4 ligase 10X Reaction Buffer* 4μl RNasin ® Ribonuclease Inhibitor (40 u/μl) 20 unit T4 kinase (9-12u/μl) 2 μl 10 mM ATP 4 μl Nuclease-Free Water to final volume 40 μl

Incubate at 37° C. for 30 minutes in a PCR thermocycler or water bath.For non-radioactive phosphorylation, use up to 300 pmol of 5′ termini ina 30 to 40 μl reaction containing 1×T4 Polynucleotide Kinase ReactionBuffer, 1 mM ATP and 10 to 20 units of T4 Polynucleotide Kinase.Incubate at 37° C. for 30 minutes. 1×T4 DNA Ligase Reaction Buffercontains 1 mM ATP and can be substituted in non-radioactivephosphorylations. T4 Polynucleotide Kinase exhibits 100% activity inthis buffer). Fresh buffer is required for optimal activity (in olderbuffers, loss of DTT due to oxidation lowers activity.

B. Annealing Fragment1 and 2 MA-fragment 3 oligo complex:

1. Reagents and instruments:

PCR thermocycler instrument or water bath

100 μg/m1 nuclease-free albumin

100 mM MgCl2

2. Assemble the following reaction mixture in a sterile microcentrifugetube:

Acceptor RNA oligo (1E) <200 ng Donor RNA oligo (3G-2G ligated oligo)<200 ng (5′ phosphorylated oligo from step A)

Appropriate ratios are as follows: Acceptor oligo:Donor oligo (Fragment1:2 MA-Fragment 3) molar ratio should be 1:1.1 to avoid fragment 1self-ligation. MgCl₂ was added to T4 ligase buffer (50 mM Tris-HCl, pH7.8, 10 mM MgCl₂, 10 mM DTT and 1 mM ATP) to final 20 mM concentration.Add Rnase free albumin to final 5 μg/ml. The final volume should be nomore than 100 μl. The solution was heated to 70° C. for 5 min, then wascooled from 70° C. to 26° C. over 2 hours and cooled from 26° C. to 0°C. over 40 minutes. Incubate at 16° C. for 16 to 17 hours using PCRinstrument.

C. Ligation of annealed oligos

Annealed oligos <15 μl 10 mM ATP 2 μl 40% PEG 18 μl T4 ligase 10X Buffer2 μl RNasin ® Ribonuclease Inhibitor (40 u/μl) 0.5 μl T4 ligase (9-12u/μl) (NEB) 1 μl Nuclease-Free Water to final volume 40 μl

D. Precipitating tRNA fragment

After ligation, 50 μl DEPC water and 150 μl phenol: chloroform wereadded and vortexed vigorously for 30 seconds. This was then centrifugedat maximum speed in a microcentrifuge for 5 minutes at room temperature.The aqueous phase was transferred to a new microcentrifuge tube (˜100μl). To this was added 2 μl 10 mg/m1 mussel glycogen, 10 μl 3M sodiumacetate, pH 5.2. This was mixed well. Then 220 μl 95% ethanol was addedand vortexed briefly. The mixture was then frozen on dry ice for 30minutes. At this point the mixture may be stored over night at −20° C.or one may proceed. In one embodiment, the RNA should preferably not bestored in DEPC water, but in ethanol, at −20° C.

Then the samples were brought to 4° C. and centrifuged at maximum speedin a microcentrifuge for 15 minutes. The position of the pellet wasnoted and the supernatant decanted or removed by pipette. Care was takennot to disturb pellet. The pellet was then washed with 70% pre-cooledethanol twice. After removing the ethanol, the wet pellet was dried witha speed vacuum for 15 min. The dried pellet was stored at −20° C., untilthe next step.

RNA Translation

A luciferase mRNA which was modified to have the stop codoncorresponding to that recognized by the anticodon of the SATA (in thepresent case UAG) was used in a standard Promega in vitro translationkit in the recommended 1 μl of concentration 1 μg/μl. One skilled in theart will understand that indeed any mRNA fragment containing theappropriate sequences may be used.

SATA was added to the experimental tubes. Control tubes which did notcontain SATA were also prepared. The quantity of SATA used wasapproximately between 0.1 μg to 500 μg, preferably between 0.5 μg to 50μg. 1 μl of Rnasin at 40 units/ml was added. Nuclease free water wasadded to make a total volume of 50 μl.

For proteins greater than approximately 150 amino acids, the amount oftRNA may need to be supplemented. For example, approximately 10-200 μgof tRNA may be added. In general, the quantity of the SATA should behigh enough to effectively suppress stop or pseudo stop codons. Thequantity of the native tRNA must be high enough to out compete the SATAwhich does not undergo dynamic proofreading under the action ofelongation factors.

Each tube was immediately capped, parafilmed and incubated for thetranslation reactions at 30° C. for 90 minutes. The contents of eachreaction tube was transferred into a 50 μl quartz capillary tube bycapillary action. The SATA was crosslinked with mRNA by illuminating thecontents of each tube with 2-10 J/cm2˜350 nm wavelength light, as perGasparro et al. (Photochem. Photobiol. 57:1007 (1993), hereinincorporated by reference). Following photocrosslinking, the contents ofeach tube were transferred into a new snapcap microfuge tube. Theribosomes were dissociated by chelating the calcium cations by adding 2μl of 10 mM EDTA to each tube. Between each step, each tube was gentlymixed by stirring each component with a pipette tip upon addition.

The optimal RNA for a translation was determined prior to performingdefinitive experiments. Serial dilutions may be required to find theoptimal concentration of mRNA between 5-20 μg/ml.

SDS-Page electrophoresis was performed on each sample, as describedabove. Autoradiography on the gel was performed, as described bySambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed.,Coldspring Harbor Press (1989), herein incorporated by reference.

The above example is instructive for the production and use of SATA(puromycin on tRNA and crosslinker on tRNA) and for the production anduse of Linking tRNA Analog (no puromycin, with crosslinker on tRNA).

Example 4: Production of a tRNA Analog Using Ribonucleotides Modified toForm Crosslinkers: Use of Psoralen and Non-Psoralen Cross-Linkers

As described above, pseudouridine can be used in some embodiments tominimize the formation of unwanted monoadducts and crosslinks. In oneembodiment, a crosslinker modified mononucleotide is formed and used.One advantage of the crosslinker modified mononucleotide is that itminimizes the formation of undesirable monoadducts and crosslinks.

As discussed above, one skilled in the art will appreciate that theSATA, Linking tRNA Analog, and Nonsense Suppressor Analog can beproduced in a number of different ways. In a preferred embodiment,psoralenated uridine 5′ mononucleotide, 2-thiocytosine, 2-thiouridine,4-thiouridine 5-iodocytosine, 5-iodouridine, 5-bromouridine or2-chloroadenosine can be produced or purchased and enzymatically ligatedto an oligonucleotide to be incorporated into a tRNA analog. Arylazides, and analogues of aryl azides, and any modifications thereto, canalso be used in several embodiments, as a linking moiety or agent. Thefollowing protocol can be employed for crosslinkers that are located onthe tRNA. One skilled in the art will understand that this protocol canalso be used for crosslinkers located on the mRNA. Thus, the followingexample is instructive on the production and use of SATA, Linking tRNAAnalog, and Nonsense Suppressor Analog.

Production of Modified Nucleotide

4-thioU, 5-iodo and 5-bromo U with and without puromycin can bepurchased already incorporated into a custom nucleotide up to 80basepairs in length (Dharmacon, Inc). Therefore, the SATA, and theLinking tRNA Analog with these crosslinkers already in place, andsimilar crosslinkers, can be purchased directly from Dharmacon, Inc.Nonsense Suppressor Analog can also be purchased from Dharmacon, Inc.

2-thiocytosine, 2-thiouridine, 4thiouridine 5-iodocytosine,5-iodouridine, 5-bromouridine or 2-chloroadenosine can all be purchasedfor crosslinking from Ambion, Inc. for the use in the Ambion MODIscriptkit for incorporation into RNA. Therefore, the SATA and the Linking tRNAAnalog along with these crosslinkers, and similar crosslinkers, can bepurchased directly from Ambion, Inc

The PO₄U_(psoralen) can be produced as follows:

(SEQ ID NO: 20) AUAUAUAUAUAUAUAUAUAUGGGGGG (seq A1) (available fromDharmacon, Inc.) (SEQ ID NO: 21) CCCCCCATATATATATATATATATAT (seq A2)(available from University of Southern California services).

The formation of a furan-sided psoralen monoadduct with the targeturidine is performed as follows:

A reaction buffer is prepared. The reaction buffer, with a pH of 7.0,contains 25 mM Tris HCL, 100 mM NaCl, and 0.32 mM EDTA.

4′hydroxy methyl-4,5′,8′-triethyl psoralen (HMT) is then added to afinal concentration of 0.32 mM and equimolar amounts of seq A1 and seqA2 are added to a final molar ratio of seq A1:seq A2:psoralen=1:1:1000.A total volume of 100 μl is irradiated at a time.

The mixture of complementary oligos, HMT, trimethylpsoralen is processedas follows: 1) Heat to 85° C. for 60 sec followed by cooling to 4° C.over 15 min, using PCR thermocycler; and 2) Irradiate for 20 to 60 minat 4° C., in Eppendorf UVette plastic cuvette, covered top withparafilm, in an RPR-200 Rayonet Chamber Reactor equipped with a coolingfan and 419 nm wave. This is either placed on an ice water bath or in a−20° C. freezer.

Steps 1 and 2 above are repeated 4 times to re-intercalate and irradiateHMT. After 4 cycles of irradiation, the free psoralens are extractedwith chloroform and all oligos (labeled and unlabeled) are precipitatedwith ethanol overnight (see precipitation step). A small aliquot issaved for gel identification.

Comparable sequences can be produced using the Ambion, Inc kit fornon-psoralen crosslinkers.

RNase H Digestion of RNAs in DNA/RNA Duplexes

The following steps are performed: (1) Dry down oligos in speed vac; (2)Resuspend pellet in 10 μL 1×Hyb Mix; (3) Heat at 68° C. for 10 minutes;(4) Cool slowly to 30° C. Pulse spin down; (5) Add 10 μL 2×RNase HBuffer. Mix. (6) Incubate at 30° C. for 60 minutes; (7) Add 130 μL StopMix.

For the Phenol/Chloroform extract: (1) Add 1 vol. phenol/chloroform; (2)Vortex well; (3) Spin down 2 minutes in room temperature microfuge; (4)Remove top layer to new tube.

For the Chloroform extract: (1) Add 1 vol. chloroform; (2) Vortex well;(3) Spin down 2 minutes in room temperature microfuge; (4) Remove toplayer to new tube.

Then, (1) Add 375 μL 100% ethanol; (2) Freeze at −80° C.; (3) Spin down10 minutes in room temperature microfuge; (4) Wash pellet with 70%ethanol; (5) Resuspend in 10 μL loading dye; (6) Heat at 100° C. for 3minutes immediately before loading.

Purification of monoribonucleotides nucleotides from the longer cDNA aswell as longer RNA fragments, is accomplished using anion exchange HPLC.The psoralen-monoadducted mononucleotides (PO₄U_(psoralen)) are thenseparated by reverse phase HPLC from mononucleotides that were notmonoadducted (PO₄U and PO₄A).

Similar digestion techniques and nucleotide incorporation, describedbelow, can also be used for non-psoralen crosslinkers using the Ambion,Inc kit.

Incorporation of Light Sensitive Nucleotides into the tRNA ComponentOligoribonuleotides

The following protocol can be used for incorporating a p U_(crosslinker)into a CUA stop anticodon. However, one skilled in the art willunderstand that other nucleotides can also be used to produce other stopanticodons and pseudo stop anticodons in accordance with the methodsdescribed herein

Generally, methods adapted from the protocols for T4 RNA ligase areused, but with some modification because of the lack of protection ofthe 3′ OH of the modified nucleotides.

5′OH CUC OH 3′ oligoribonucleotides (seq B1) can be purchased fromDharmacon, Inc. and can be as acceptors in the ligation. The molar ratioof B1 to psoralenated mononucleotides is preferably kept at 10:1 to 50:1so that the modified U's will be greatly out-numbered, therebypreventing the formation of CUC(U_(crosslinker))_(N). This makes one ofthe preferred reactions:CUC+

CUCU _(psoralen)

In one embodiment, the product is purified by sequential anion exchangeand reversed phase HPLC to ensure that the psoralenated U and the longerpsoralenated 7 mer are separated. The 7 mer is then 3′ protected byligation with pAp yielding CUCU_(crosslinker)AP (Fragment 2B).

This is again purified with anion exchange HPLCF or the next ligation.

First Ligation of Fragment 2B to 1B or 1B1

This 2B fragment can be used in a tRNA analog that has a stable acceptoror one that has a native esterified acceptor. In one embodiment, toassure that the native 3′ end can be aminoacylated by native AA-tRNAsynthetases, the acceptor stem is modified in that version of theanalog. In the SATA version, in one embodiment, the 3′ fragment ismaintained with a commercially prepared puromycin as the acceptor. Thus,in one embodiment, the following are used in two different 5′ ends:

(SEQ ID NO: 22) 5′ OHGCGGAUUUAGCUCAGUUGGGAGAGCGCCAGA 3′ seq 1B (to beused with the tRNA analog with the stable puromycin acceptor) and (SEQID NO: 23) 5′ OHGGGGCUUUAGCUCAGUUGGGAGAGCGCCAGA 3′ seq 1B₁ (to be usedwith the native esterified acceptor).

The ligation is performed again with T4 RNA ligase and purified bylength. The equation for sequence 1B is as follows:

(SEQ ID NO: 22) 5′OHGCGGAUUUAGCUCAGUUGGGAGAGCGCCAGA 3′ +CUCU_(crosslinker)APO₄ 3′ 

(SEQ ID NO: 24) 5′ OHGCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACUCU_(crosslinker)APO₄ 3′ For sequence 1B₁: (SEQ ID NO: 23)5′ OHGGGGCUUUAGCUCAGUUGGGAGAGCGCCAGA + CUCU_(crosslinker)APO₄ 3′ 

(SEQ ID NO: 25) 5′ OHGGGGCUUUAGCUCAGUUGGGAGAGCGCCAGACUCU_(crosslinker)APO₄ 3′

Ligation of the Two Half-Molecules of the TRNA Analog

The above product is treated with T4 polynucleotide kinase in twoseparate steps to remove the 3′ phosphate and add a 5′ phosphate.

The newly prepared 5′ and 3′ half molecules ends are then ligatedgenerally following the previous protocols. The 3′ sequencescorresponding to the respective 5′ sequences are as follows:

Sequence 1B: (Ψ = pseudouridine) (SEQ ID NO: 24)5′ PO₄GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACUCU_(crosslinker) A3′ corresponded to the 3′ half: (SEQ ID NO: 31)5′PO₄UGGAGGUCCUGUGTΨCGAUCCACAGAAUUCGCACCPur 3′, 3B and sequence 1B1,(SEQ ID NO: 25) 5′ OHGGGGCUUUAGCUCAGUUGGGAGAGCGCCAGACUCU_(crosslinker)APO₄  corresponded to 3′ half (SEQ ID NO: 32)5′PO₄UGGAGGUCCUGUGTΨCGAUCCACAGAAUCUCCACCA3′.

The latter is recognizable by the aminoacyl tRNA synthetase for alaninein E. coli.

The example described above can be used to make and use the SATA,Linking tRNA, and the Nonsense Suppressor tRNA.

Example 5: Placement of Crosslinkers on the mRNA for SATA and NonsenseSuppressor tRNA

In several embodiments, the crosslinker (such as psoralen or anon-psoralen crosslinker) is not placed on the tRNA, but rather locatedon the mRNA. For example, in one embodiment, the SATA comprises apuromycin located on the tRNA, while the crosslinker is on the mRNA. Inyet another embodiment, the Nonsense Suppressor tRNA is used, and thiscomprises a tRNA with no puromycin, with the crosslinker being on themRNA. Placement of the crosslinker on the message (the mRNA) can beaccomplished as set forth below. The relevant sequence is as follows:

(SEQ ID NO: 26) GGGUUAACUUUAGAAGGAGGUCGCCACCAUG GUU AAA AUG AAA AUG AAAAUG AAA AUG U_(crosslinker)AG

For convenience only, and in one embodiment, a message with both Kozakand Shine Dalgarno sequences that has a large number of methioninecodons for ³⁵S labeling is used.

For 4-thiouridine, 5-bromouridine and 5-iodouridine, the message can bepurchased fully-made from Dharmacon, Inc. For aryl azides, the methodrecited in Demeshkina, N, et al., RNA 6:1727-1736, 2000, hereinincorporated by reference, can be used.

For 2-thiocytosine, 2-thiouridine, 5-iodocytosine, or 2-chloroadenosine,the modified bases can be purchased as the 5′ monophosphate nucleotidefrom Ambion, Inc. When psoralen is used as the crosslinker, the modified5′ monophosphate nucleotide is made as above.

The modified 5′ monophosphate nucleotides are first incorporated intohexamers to facilitate purification. The construction of uridinecontaining crosslinkers is shown but in several embodiments, the otherbases can be incorporated into both stop and pseudo stop codons usingsimilar techniques:

AUG+pUcrosslinker

AUGUcrosslinker was accomplished using a similar protocol describedabove, except a preponderance of AUG was used because of the absence ofa 3′ protection of the pNcrosslinker. The product was purified by anionexchange HPLC from the excess of AUG. Then 5′ pAGbiotin 3′ was addedwith T4 RNA ligase. The 3′ biotin was simply a convenient 3′ blockinggroup available form Dharmacon. The resultingAUGU_(crosslinker)AG_(biotin) was again purified followed by 5′phosphorylation and ligated to:

(SEQ ID NO: 27) GGGUUAACUUUAGAAGGAGGUCGCCACCAUGGUUAAAAUGAAAAUGAAAA UGAAA(sequence M1) to produce (SEQ ID NO: 28)GGGUUAACUUUAGAAGGAGGUCGCCACCAUGGNNAAAAUGAAAAUGAAAAUGAAAAUGU_(crosslinker)AG_(biotin).

The yield is high enough to obviate purification. Accordingly, using theprotocol described above, SATAs and Nonsense Suppressor tRNAs can bemade and used in accordance with several embodiments of the presentinvention.

Example 6: Using tRNA Systems that Do Not Require Puromycin

Several embodiments of the present invention provide a system and methodthat do not require puromycin, puromycin analogs, or other amidelinkers. In one embodiment, Linking tRNA Analogs and Nonsense SuppressortRNAs do not require puromycin and can be made and used according to thefollowing example.

For systems without puromycin, a translation system to aminoacylate thetRNA can be used. In other embodiments, aminoacylation can beaccomplished chemically. One skilled in the art will understand how tochemically aminoacylate tRNA. Where translation systems are used, anytype of translation system for aminoacylation can be employed, such asin vitro, in vivo and in situ. In one embodiment, am E-coli translationsystem is used. An E. coli translation system is used for systems with atRNA modified to be recognized by the aaRS^(Ala). In one embodiment,this is preferable for systems without the stable acceptor (e.g. thepuromycin)

3 mcg of each of the following mRNA's are translated in 40 microliterseach of Promega S30 E. coli translation mixture:

a) (SEQ ID NO: 28) GGGUUAACUUUAGAAGGAGGUCGCCACCAUG GUU AAA AUG AAA AUGAAA AUG AAA AUGUcrosslinkerAGbiotin and b) (SEQ ID NO: 29)GGGUUAACUUUAGAAGGAGGUCGCCACCAUG GUU AAA AUG AAA AUG AAA AUG AAA AUGUAG

3 mcg of amber suppressor tRNA manufactured as above are added to thefirst. 3 mcg of suppressor with crosslinker on the anticodon are addedto the second. 35S-methionine is added to both and the mixtures are thenincubated at 37° C. for 30 minutes. The reactions are then rapidlycooled by placement in an ice bath, transferred to a flat Petri dish andfloated in an ice bath so that the mixture is 1.5 cm below a ˜350 nmlight source. They are exposed at ˜20 J/cm for 15 min.

After irradiation, the mixtures are phenol extracted and ethanolprecipitated. In this manner, systems such as the Linking tRNA Analogsand Nonsense Suppressor tRNAs are aminoacylated and used to connect themessage (mRNA) to its coded peptide in accordance with severalembodiments of the present invention.

Example 7: Alternative Sequences

In a preferred embodiment, Fragments 1, 2 and 3, described above inExample 1, have the following alternate sequences:

Fragment 1 (SEQ ID NO: 13)

5′ PO4 GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGA N3-Methyl-U 3′

Fragment 2 (SEQ ID NO: 14)

5′ UCUAAGΨCΨGGAGG 3′

Fragment 3—Unchanged from the Sequence Listed Above (SEQ ID NO: 6)

5′ PO4 UCCUGUGTΨCGAUCCACAGAAUUCGCACC Puromycin 3′

Using the methods described above, the sequence of alternative Fragments1+2+3 was (SEQ ID NO: 15):

5′PO4GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGA(N3-MethylU)UCUPsoralenAAGΨCΨGGAGGUCCUGUGTYCGAUCCACAGAAUUCG Puromycin 3′

For linking tRNA Analog and Nonsense Suppressor tRNA, the abovesequences are similar, except adenosine is used to replace puromycin.

While a number of preferred embodiments of the current invention andvariations thereof have been described in detail, other modificationsand methods of use will be readily apparent to those of skill in theart. For all of the embodiments described above, the steps of themethods need not be performed sequentially. Accordingly, it should beunderstood that various applications, modifications and substitutionsmay be made without departing from the spirit of the invention or thescope of the claims.

What is claimed is:
 1. A system for making targeted therapeutics, thesystem comprising: a first protein-mRNA cognate pair comprising a firstmRNA sequence linked to a first protein by a first stable aminoacyl tRNAanalog (SATA), the first protein encoded by the first mRNA sequence, thefirst protein capable of specifically interacting with an agent ofinterest; and a second protein-mRNA cognate pair comprising a secondmRNA sequence linked to and a second protein by a second SATA, thesecond protein encoded by the second mRNA and capable of binding humanIgG, wherein each one of the first and second SATA is modified with apuromycin moiety such that the each one of the first and second SATAmimics an aminoacyl-Tyr tRNA.
 2. The system of claim 1, furthercomprising a fused protein comprising said first protein linked withsaid second protein.
 3. The system of claim 2, wherein a first portionof said fused protein is capable of interacting with said agent ofinterest and a second portion of said fused protein is capable ofbinding human IgG.
 4. The system of claim 1, wherein said agent ofinterest targets one or more of the blood, blood vessels, nervoustissue, muscle tissue, and one or more ion channels.
 5. The system ofclaim 1, wherein said agent of interest induces muscle paralysis, orprevents blood clotting, or induces increased gastrointestinal watersecretion.
 6. The system of claim 3, wherein said second portion is anon-antibody protein.
 7. The system of claim 1, wherein said secondportion is capable of binding to the heavy chain of an antibody.
 8. Thesystem of claim 7, wherein said second portion is capable of binding tothe constant region of the heavy chain of said antibody.
 9. The systemof claim 7, wherein said second portion is capable of binding to the CH1region of the heavy chain.
 10. The system of claim 1, wherein whereinagent of interest is selected from the group consisting of animaltoxins, insect toxins, plant toxins, algae-derived toxins, fungi-derivedtoxins, bacterial-derived toxins, biowarfare agents, and biopathwaymodulators.
 11. A system for making targeted therapeutics, the systemcomprising: a first protein-mRNA cognate pair comprising a first mRNAsequence linked to a first protein by a first linker, the first proteinencoded by the first mRNA sequence, the first protein capable ofspecifically interacting with an agent of interest; and a secondprotein-mRNA cognate pair comprising a second mRNA sequence linked toand a second protein by a second linker, the second protein encoded bythe second mRNA and capable of binding human IgG, wherein each one ofthe first and second linker is selected from the group consisting of: astable aminoacyl tRNA analog (SATA) modified with a psoralen moietymonoadducted to an anticodon of the SATA, an aryl azide, an analogue ofaryl azides, a pseudouridine, a puromycin moiety, and SEQ ID NO:
 6. 12.The system of claim 11, further comprising a fused protein comprisingsaid first protein linked with said second protein.
 13. The system ofclaim 12, wherein a first portion of said fused protein is capable ofinteracting with said agent of interest and a second portion of saidfused protein is capable of binding human IgG.
 14. The system of claim13, wherein said second portion is a non-antibody protein.
 15. Thesystem of claim 11, wherein said agent of interest targets one or moreof blood, a blood vessel, a tissue of a nervous system, a muscle tissue,or one or more ion channels.
 16. The system of claim 11, wherein saidagent of interest induces muscle paralysis, or prevents blood clotting,or induces increased gastrointestinal water secretion.
 17. The system ofclaim 11, wherein the agent of interest is selected from the groupconsisting of animal toxins, insect toxins, plant toxins, algae-derivedtoxins, fungi-derived toxins, bacterial-derived toxins, biowarfareagents, and biopathway modulators.
 18. The system of claim 11, whereinsaid second portion is capable of binding to the heavy chain of anantibody.
 19. The system of claim 18, wherein said second portion iscapable of binding to the constant region of the heavy chain of saidantibody.